Nitrite and Nitrosamine Synthesis by Hepatocytes Isolated from Normal Woodchucks (Marmota Monax) and Woodchucks Chronically Infected with Woodchuck Hepatitis Virus1

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(CANCER RESEARCH 52, 4139-4 MX August 1. 1992] Nitrite and Nitrosamine Synthesis by Hepatocytes Isolated from Normal Woodchucks (Marmota monax) and Woodchucks Chronically Infected with Woodchuck Hepatitis Virus1

Rui Hai Liu, James R. Jacob, Bud C. Tennant, and Joseph H. Hotchkiss2

Institute of Comparative and Environmental Toxicology Â¡R.H. I... J. H. II.] and College of Veterinary Medicine /J. R. J., B. C. T.], Cornell University, Ithaca, New York 14853

ABSTRACT nitrate through the same pathway. Nitrite synthesis by rat hepa tocytes has also been reported; however, hepatocyterKupffer Hepatocytes isolated from woodchucks (Marmota monax) were shown to produce nitrite in vitro from i.-arginine after stimulation with cell cocultures, but not hepatocytes alone, produced nitrite, nitrate, and NO in response to LPS (15-17). lipopolysaccharide (LPS). Hepatocytes isolated from woodchucks that were chronic carriers of woodchuck hepatitis virus formed twice as much The objective of this study was to determine if nitrite was produced in hepatocytes through the i.-arginine-nitric oxide nitrite as hepatocytes from noninfected animals. Nitrite synthesis by hepatocytes was directly related to i-arginine and LPS concentrations in pathway after stimulation with LPS and without the presence of the tissue culture medium and reached a plateau at 0.5 IHM[-arginine Kupffer cells and to compare nitrite synthesis and nitrosation and 1.0 Â»ig/mlLPS. LPS-stimulated hepatocytes nitrosated morpholine capability of hepatocytes isolated from normal and WHV-in- to form /V-nitrosomorpholine in the presence of i.-arginine at a physio fected woodchucks. These data might explain our in vivo data logical pH of 7.4. There was a 10-fold increase in A'-nitrosomorpholine showing that WHV infection resulted in a higher endogenous production when hepatocytes were stimulated with LPS compared to nitrate and NDMA formation. unstimulated hepatocytes under similar conditions when both nitrite and morpholine were directly added to the medium. /V'-monomethyl-t - arginine, a selective inhibitor of nitric oxide synthase, inhibited forma MATERIALS AND METHODS tion of both nitrite and /V-nitrosomorpholine. These results demonstrate that nitrosating agents are formed in hepatocytes via the i-arginine- Reagents. NMOR and morpholine were purchased from Aldrich nitric oxide pathway. This suggests that endogenous formation of car Chemical Co. (Milwaukee, WI). LPS (E. coli, serotype 0127:B8, phenol cinogenic Y-iiifroso compounds could influence the process of hepato- extract), NDMA, NDPA, i-arginine-HCl, L-NMMA, insulin, gluca- gon, /V-l-naphthylethylenediamine dihydrochloride, sulfanilamide, and carcinogenesis in woodchucks with chronic woodchuck hepatitis virus infection. sodium azide were purchased from Sigma Chemical Co. (St Louis, MO). Minimum essential media (free of t-arginine, without phenol red). Hanks' balanced salt solution (Ca:+/Mg:+ free), Williams' Media INTRODUCTION E, fetal bovine serum, HEPES, and penicillin-streptomycin solution were purchased from G1BCO Laboratories (Grand Island, NY). Low- Woodchucks chronically infected with WHY,' which is endotoxin fetal bovine serum was obtained from HyClone Laboratories closely related to human hepatitis B virus, develop hepatocel- (Logan, UT). All water was double distilled in glass. NMOR and nitrite lular carcinoma with high frequency (1-3). We recently demon were not detected in the distilled water or reagent blanks. strated that [l5N]nitrate and [I5N]NDMA could be detected in Hepatocyte Isolation. Three normal male woodchucks weighing 4.5 woodchuck urine after administration of i.-[l5N2]arginine and Â±0.7 (SD) kg and four WHV chronic carrier male woodchucks LPS (4). This indicated that t-arginine is a precursor of both (4.3 Â±0.5 kg) were utilized in these studies. Chronic WHV carrier nitrate and the hepatocarcinogen NDMA, most probably as a woodchucks had been infected with the virus at birth as described result of the formation of nitrosating agents during the oxida previously (2, 4). Animals were tested for woodchuck hepatitis surface tion of L-arginine to NO and citrulline. Woodchucks chroni antigen as an indicator of infection status. Hepatocyte cultures derived from WHV chronic carrier woodchucks were also monitored for WHV cally infected with WHV formed more nitrate and NDMA than surface antigen secretion by an enzyme-linked immunosorbent assay. control animals which suggested that endogenous production of The WHV-infected cultures remained surface antigen positive through jV-nitroso compounds including NDMA may represent an eti- out the culture period, indicating continued viral expression. Hepato ological factor in woodchuck hepatocarcinogenesis (5). cyte isolation and culture were by modification of a previous described Macrophages in culture, when stimulated with LPS from procedure (18). Animals were anesthetized with pcntobarbital and the Escherichia coli, produce nitrite, nitrate, and nitrosating agents left lateral lobe of the liver was removed surgically. The liver lobe was from L-arginine (6-10). The guanidino nitrogen of L-arginine is perfused with Hanks' balanced salt solution [Ca2+/Mg-* free, with 10 ITIMHEPES, 1.0 MIMethylenebis(oxyethylenenitrilo)tetraacetic acid, the source of the nitrogen incorporated into NO, with the fur pH 7.4] at 37Â°Cfor 10 min, followed by perfusion with 0.06% collage- ther oxidation of NO to nitrite and nitrate in vitro (7, 8). Other nase D (Boehringer Mannheim Biochemical, Indianapolis, IN) at 37Â°C cell types including endothelial cells (11, 12), platelets (13), and for 20 min. The liver capsule was removed and hepatocytes were dis neurons (14) have been reported to produce NO, nitrite, and sociated by gentle agitation. The cell suspension was Tillered through two layers of gauze pads into 5% fetal bovine serum/Williams' Media E (4Â°C),followed by centrifugation at 50 x g for 4 min at 4'C. The Received 2/18/92; accepted 5/20/92. The costs of publication of this article were defrayed in part by the payment of resulting hepatocytes were washed and pelleted for three additional page charges. This article must therefore be hereby marked advertisement in accord cycles to achieve an hepatocyte purity of >96%. The cells were enu ance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This research was supported in part by CA-37264 and NO1-A1-82698 from the merated and viability was >85% as determined by trypan blue exclu U.S. Public Health Service, and by NIH/NIAID National Research Service Award sion. Nonparenchymal cell (endothelial cells, Kupffer cells, and fat- F32 AI0834I and American Liver Foundation Research Fellowship. storing cells) content was found to be <4% by Giemsa and Wright's 2 To whom requests for reprints should be addressed. 'The abbreviations used are: WHV, woodchuck hepatatis virus: LPS, li stains on initial cell isolations and cell cultures 3 days postplating popolysaccharide; L-NMMA, iV(i-monomethyl-i-arginine; NDMA. ,V-nitrosodi- cytospin smear. Cell cultures were also Giemsa stained and counted methylamine; NMOR. A'-nitrosomorpholine; NDPA, A'-nitrosodi-n-propylamine; before monolayer formation. All tests indicated a hepatocyte purity of HEPES. 4-(2-hydroxycthyl)-l-pipera7.ineethanesulfonic acid. >96% and it was assumed that less than 40% of the maximum 4% 4139

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. HEPATOCYTES AND IN I'lTRO NITRITE AND A'-NITROSAMINE FORMATION contaminant were Kupffer cells. Hepatocytes were plated on collagen- coated flasks or wells at a density of 1 x IO5cells/cm2 and incubated for [ 1 noL-AriN\1 adherence at 37Â°Cand 5% CO2. After 2.5 h incubation, nonadherent SO L-ArjtWI cells were removed by washing. Adherent cell monolayers were incu L-NUIU-1â€¢\L-Arg +â€¢ bated with serum-free medium supplemented with growth factors and hormones as described by Lanford et al. (18). Seventy-two h after plat â€¢¿*-C 40 ing, the hepatocyte monolayers were washed with phosphate-buffered CM saline, followed by cultivation under the appropriate experimental con ditions. In all experiments, the medium consisted of minimum essential (J 30 media (i -arginine free, phenol red free) supplemented with 5% low- e endotoxin fetal bovine serum (HyClone, Logan, UT), insulin 5 jig/ml, O 10 m\i HEPES, penicillin 50 units/ml, and streptomycin 50 ng/ml; 2 in imi L-arginine, 1 mv L-NMMA, 10 Mg/mlLPS, and 10 mm morpholine CN 20 were added as indicated. The primary hepatocytes exhibited typical cuboidal cell geometry, containing a small nucleus, often binucleated, \ surrounded by a granular cytoplasm, characteristic of a parenchyma! 10 s cell type. \N ss\ The rat hepatocytes were isolated from two adult female Long-Evans \\ rats (277 Â±12 g). This procedure was identical to that for hepatocyte ^ f*T^L^:s isolation from woodchucks except that a 0.03% collagenase solution was utilized. LPS (-) LPS (-O LPS (-) LPS Nitrite Analysis. Nitrite was quantified in cell culture supernatants by colorimetrie assay based on Griess reaction (19). Supernatant fluid (100 M!)was added to an equal volume of Griess reagent in microtiter Control WHV-Carrier plates. Absorbance at 550 nm was measured using a microplate reader Fig. 1. Nitrite synthesis by hepatocytes isolated from WHV carriers and nor (Bio-Tek EL3I2) and nitrite values were calculated from a standard mal woodchucks. Hepatocytes (2.5 x IO6)were cultured for 24 h in 2 ml medium with (+) or without (â€”)2min L-arginine, 10 nu ml LPS, and 1 mm L-NMMA as curve after correcting for background. indicated. Bars, SD; n = 3 animals for controls; n = 4 animals for WHV carriers; NMOR Analysis. Sodium azide was added to samples used for duplicate cultures were derived from each animal). NMOR analysis (final concentration, 0.01 M)as a nitrosation inhibitor. A 1-ml aliquot of supernatant was mixed with l g muffled Celile (1:1, w/v) and 35 ng NDPA and packed into a 10- x 100-mm glass column containing 5 g anhydrous sodium sulfate and glass wool at the top and (13.06 Â±8.14 MMnitrite/2.5 x IO6 cells/24 h) and WHV bottom. MÃ©thylÃ¨nechloride(8 ml) was passed through the column and chronic carrier woodchucks (12.05 Â±13.37 MMnitrite/2.5 x 106 4 ml of eluate were collected and slowly concentrated to 0.5 ml under cells/24 h) (Fig. 1). Correcting for nitrite production in the a stream of N2. Samples were analyzed for NMOR by gas chroma- absence of L-arginine but with LPS stimulation, hepatocytes tography-Thermal Energy Analyzer (Hewlett-Packard Model 5890 from WHV chronic carrier woodchucks produced 3.4-fold GC; Model 543 TEA; 3-m x 2-mm inside diameter glass column con taining 10% Carbowax 20 M on 80-100 Chromosorb WHP). Temper more nitrite than normal hepatocytes (34.93 Â±12.5 and 10.25 atures were: initial hold at 135'C for 2.2 min; 10Â°C/minto 180Â°Cwith Â±9.7, P < 0.025, respectively). an 8-min hold; injection port, 210"C; pyrolyzer, 550Â°C;interface, L-NMMA, which inhibits the nitric oxide pathway (7), in 200"C; and cold trap, 150Â°C.Peak heights were compared with external hibited nitrite production by hepatocytes stimulated with LPS NMOR and NDPA standards. Recovery of NMOR (50 ng) and NDPA (Fig. 1). Indomethacin also inhibited nitrite production by (internal standard, 35 ng) from spiked medium averaged 99.5 and 99%, hepatocytes (Fig. 2). Inhibition by indomethacin was observed respectively. Addition of dimethylmorpholine to several samples did at a threshold concentration of 0.2 MM.At higher concentra not result in the detectable formation of yV-nitrosodimethylmorpholine. tions, inhibition of nitrite synthesis increased with increasing indomethacin concentration and at an indomethacin concen RESULTS tration of 1.0 MM,there was 94% inhibition of nitrite synthesis. The amount of nitrite produced by woodchuck hepatocytes LPS-induced Nitrite Synthesis by Hepatocytes. Stimulation was directly related to the dose of LPS and L-arginine added to of woodchuck hepatocytes in culture with LPS resulted in the the medium (Figs. 3 and 4). Nitrite synthesis was stimulated by detection of significant amounts of nitrite when L-arginine was as little as 0.01 Mg/rnl LPS and began to plateau at a LPS present in the medium (Fig. 1). Hepatocytes isolated from con concentration of >1.0 Mg/ml (Fig. 3). Nitrite production by trol animals in the presence of LPS and L-arginine formed 23.3 woodchuck hepatocyte-treated LPS increased with increasing Â±7.61 MMnitrite/2.5 X IO6 cells/24 h, which was a 12.5-fold L-arginine concentration and leveled off at an L-arginine con increase over unstimulated cells [P< 0.01 (Fig. 1)]. Hepatocytes centration of 0.5 HIML-arginine (Fig. 4). isolated from WHY chronic carrier woodchucks, following Hepatocytes isolated from Long-Evans rats formed 30.57 MM LPS stimulation and L-arginine addition, formed 46.98 Â±25.8 nitrite/2.5 x IO6 cells/24 h after stimulation by 10 Mg/ml LPS MMnitrite/2.5 X IO6 cells/24 h, which was 25-fold more nitrite and 2 HIML-arginine (Fig. 5). Without added L-arginine, rat than unstimulated hepatocytes in the presence of L-arginine hepatocytes produced 16.7 MMnitrite/2.5 x IO6 cells/24 h when (P < 0.01). Hepatocytes isolated from WHV chronic carrier cultured with LPS. L-NMMA also inhibited nitrite synthesis by woodchucks produced significantly (Mann-Whitney test, P < rat hepatocytes. 0.02) more nitrite than normal hepatocytes after stimulation Hepatocyte-mediated Nitrosation. LPS-stimulated hepato by LPS in the presence of L-arginine. Hepatocytes from both cytes nitrosated added morpholine to form NMOR in the pres control and WHV carrier woodchucks produced only small ence of L-arginine under physiological conditions (pH 7.4, amounts of nitrite without LPS stimulation (Fig. 1). Without 37Â°C;Table 1). LPS-stimulated hepatocytes produced signifi the addition of L-arginine, hepatocytes produced 6-7-fold more cantly more NMOR (336 nivt) than unstimulated hepatocytes nitrite with LPS stimulation than without LPS in both normal (55.5 HIM)whenthe latter were incubated with 93.6 MMnitrite for 4140

100 DISCUSSION Nitrite synthesis by rat hepatocyterKupffer celi cocultures, but not hepatocytes alone, has been reported. Kupffer celhhepa- tocyte cocultures produced more nitrite than Kupffer cells alone Â¡nresponse to LPS. Hepatocytes exposed to medium trans ferred from LPS-stimulated Kupffer cells also produced nitrite and nitrate (15-17). We found that neither woodchuck nor rat hepatocytes re quired the presence of Kupffer cells or medium transferred from LPS-stimulated Kupffer cells to produce nitrite. The reasons that our results differ from the previously reported requirement

J3 N n "oj 0.0 0.2 0.4 0.6 0.8 U

Indomethacin Cone. M Fig. 2. Inhibition of nitrite synthesis in woodchuck hepatocytes by indomctha- IO cin. Hepatocytes(1.5 x IO6) were cultured for 24 h in I ml medium containing 2 HIML-arginine and 10 pg/ml LPS (hepatocytes collected from single animal).

12.5

A CM 10.0 n

B 7.5 O Â«H L-Arg Conc (mM) M Fig. 4. Response of nitrite production by woodchuck hepatocytes as a function in of [.-arginine concentration. Hepatocytes (1.5 x IO6) were cultured for 24 h in 2 5.0 ml medium containing 10 us/ml LPS and increasing amounts of i-arginine. Bars, SD; n = 2 animals; duplicate cultures were derived from each animal.

2.5 50

0.0 CM 40 10 20 30 40 50 "5-2 o LPS Fig. 3. Nitrite production by woodchuck hepatocytes as a function of LPS 30 concentration. Hepatocytes (1.5 x IO6) were cultured for 24 h in 2 ml medium containing 2 mM i.-arginine and increasing amounts of LPS. Bars, SD; n = 4 animals; duplicate cultures were derived from each animal. 20

72 h. This amount of nitrite (93.6 n\\) is greater than that found in the medium from stimulated hepatocytes. LPS treatment induced 10-fold greater nitrosation activity than untreated 10 hepatocytes when corrected for background NMOR formation. The yield of NMOR from stimulated hepatocytes represented 0.6% of the total nitrite produced in culture and 0.003% of the morpholine added to the medium. L-NMMA not only inhibited formation of nitrite but also inhibited A'-nitrosation of morpho no l. Arg L-Arg L-Ar| t L-NUUA Fig. 5. Nitrite production by rat hepatocytes. Rat hepatocytes (2.5 x 10*) were line in culture, indicating that the nitrosating agents were de cultured for 24 h in 2 ml medium containing 10 pg/ml LPS. 2 HIMi-arginine, and rived from the i.-arginine-nitric oxide pathway in hepatocytes. I mM L-NMMA as indicated. Bars, SD; n = 2 animals. 4141

Table 1 N-.V/'frojaf/on of morpholine by woodchuck hepatocytes produce nitrite from L-arginine through a similar pathway to Harvest macrophages. Preliminary experiments indicated that 4-di- methylaminoantipyrine inhibited nitrite production but exhib Cell+ IHM)+ MM)+L-NMMA(1IHM)+NMOR(IM)336 ited significant cell toxicity. Indomethacin is used clinically as Â±24Â° Â±4.8 a nonsteroidal antiinflammatory and antipyretic agent. Indo + ++MOR(IO + 18.5 Â±3.8 2.27 Â±0.28 methacin also inhibited, in a concentration-dependent manner, +LPS(lOng/ml)+ + 55.5 Â±8.75 79.0 Â±0.8 +NO7(94 25.7 Â±3.35NOj(MM)55.10.85 Â±0.29 LPS-induced production of nitrite by hepatocytes with lower â€¢¿MeanÂ±SD. toxicity (Fig. 2). The inhibitory activity was observed at a threshold concentration of 0.2 MM,which is lower than found in plasma (2.8 MM) following therapeutic administration (23). for Kupffer cells or extracts are unknown. In our hands, hepa These data suggest that antiinflammatory drugs may also in tocytes isolated from Long-Evans rats produced 30.57 MM hibit NO synthetase. nitrite/2.5 X IO6 cells/24 h from i-arginine with stimulation by Hepatocytes from WHY chronic carrier woodchucks pro LPS (Fig. 5). In the absence of L-arginine, rat hepatocytes pro duced twice the amount of nitrite from L-arginine as hepato duced 16.9 MMnitrite/2.5 X IO6 cells/24 h when stimulated by cytes from normal animals after stimulation by LPS. These LPS. These results in rats are similar to our results in wood- results are consistent with those obtained in vivo (4) in which chucks, thus excluding species difference. unstimulated WHV-infected animals excreted 3-fold more ni The purity of our hepatocytes was >96% which is comparable trate than control animals. Treatment of WHV-infected ani to the >95% reported by Curran et al. (16). In addition, Billiar mals with LPS increased nitrate excretion 15-fold, while unin- et al. (15) used a Kupffer cell:hepatocyte ratio of 5:1 in their fected animals increased nitrate excretion 4-fold. cocultures. Therefore, the high concentration of nitrite pro The finding that LPS-stimulated hepatocytes form N-nitros- duced in our >96% purity hepatocyte cultures could not be due amines under physiological conditions (pH 7.4) when morpho to contamination with other cell types. line was added to the culture medium is similar to the results of We also tested different matrices to which cells were at Miwa et al. (9), utilizing immunostimulated macrophages. tached. There was no difference between collagen-coated and When nitrite was added to unstimulated hepatocytes at a con gelatin-coated matrices (data not shown). Initially, our hepato centration higher than that found in LPS-stimulated cultures cytes were incubated with serum-free medium. Later we tested after 72 h of incubation, only a small amount of NMOR (55.5 the effect of culture medium and found that hepatocytes incu nivi)was observed. Significantly higher NMOR concentrations bated with conventional medium produced more nitrite than (336 n\i) were observed during stimulation with LPS. lyengar et the hepatocytes incubated with serum-free medium (data not al. (8) also reported that when L-[l5N2]arginine was present in show). We have recently developed an immortalized woodchuck hepatocyte cell line which produces nitrite from L-argin the medium, the enrichment of labeled nitrogen in NMOR was virtually identical to that in nitrate and nitrite. L-NMMA, ine upon stimulation by LPS. Data from these immortalized which selectively inhibits the NO pathway (7), also inhibited cells further support our observation that hepatocytes form NO and nitrite from L-arginine after stimulation by LPS without the NMOR formation in our experiments, which indicated that the nitrosating agents were from the L-arginine-nitric oxide path requirement for Kupffer cells. Hepatocyte cultures which did not contain added i -arginine way in hepatocytes. These in vitro data support our observation that the L-arginine-nitric oxide pathway results in the in vivo produced 6-7-fold more nitrite after LPS stimulation than without LPS. Macrophages and endothelial cells have been formation of NDMA (4). reported not to form significant amounts of nitrite without These results indicate that formation of NMOR is not due to L-arginine addition (6, 8, 12, 20). Although an i.-arginine pool acid-catalyzed aqueous nitrosation via nitrite, but rather to a nonacidic, cell-mediated, neutral nitrosation via NO, which is could make a partial contribution, formation of this small amount of nitrite without extracellular L-arginine may be due to an intermediate in the L-arginine to nitrite/nitrate pathway synthesis of L-arginine from L-citrulline via the urea cycle (21). (8, 9, 24). LPS-stimulated hepatocytes convert the guanido ni This L-arginine could then serve as a substrate for NO syn- trogen of L-arginine into NO. NO reacts with dissolved oxygen thetase in hepatocytes. (O2) to produce NO2, which exists in equilibrium with the Nitrite formation increased with the increasing amount of potent nitrosating agents N2O3 and N2O4. Both of these inter LPS in the medium (Fig. 3). The dose-response curve for the mediates are capable of nitrosating dialkylamines in neutral LPS-induced nitrite synthesis by hepatocytes was similar to aqueous solution to form nitrosamines or reacting with water to that observed in macrophages (22). Nitrite synthesis by hepa yield nitrite and nitrate (7, 8, 25). tocytes was initiated by as little as 0.01 Mg/ml LPS and leveled Our data indicate that hepatocytes produce nitrite in vitro off at a dose of >1.0 Mg/ml (Fig. 3). 7-Interferon has been from i.-arginine when stimulated by LPS and that WHY reported to be required or to be synergistic with LPS in trig chronic carrier woodchucks form significantly more nitrite than gering nitrite synthesis by macrophages (22). LPS alone, how normal hepatocytes. Hepatocytes also form NMOR under ever, was sufficient to induce nitrite synthesis by hepatocytes physiological conditions (pH 7.4). This indicates that NO for under our experimental conditions. mation from L-arginine in response to immunostimulation re Nitrite synthesis after LPS stimulation was dose related to sults in the endogenous formation of hepatocarcinogenic /V-ni- the concentration of L-arginine (Fig. 4). Nitrite formation lev troso compounds and that such compounds could have an eled off at a concentration of 0.5 miviL-arginine. lyengar et al. etiological role in hepatocarcinogenesis. This further supports (8) confirmed that i.-arginine is the source of the nitrogen for our speculation that chronic hepatitis could increase the risk of nitrite using [l5N]-labeled L-arginine. L-NMMA, which selec liver carcinoma by stimulating the NO pathway (4). Nitric ox tively inhibits the NO pathway, inhibited nitrite production by ide has recently been shown to cause deamination of DNA and woodchuck hepatocytes (Fig. 1), indicating that hepatocytes has also been shown to be mutagenic (26). 4142

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 1992 American Association for Cancer Research. Nitrite and Nitrosamine Synthesis by Hepatocytes Isolated from Normal Woodchucks ( Marmota monax) and Woodchucks Chronically Infected with Woodchuck Hepatitis Virus

Rui Hai Liu, James R. Jacob, Bud C. Tennant, et al.

Cancer Res 1992;52:4139-4143.

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