1Department of Physiology, Medical College of Georgia, Augusta, Georgia 30912; and 2Department of Pharmacology, New York Medical College, Valhalla, New York 10595
Submitted 14 February 2003 ; accepted in final form 4 April 2003
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ABSTRACT |
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cytochrome P-450 4A; NG-nitro-L-arginine methyl ester
Normal pregnancy in humans and rats is associated with increases in glomerular filtration rate and renal blood flow (21) along with significant decreases in arterial pressure and total peripheral resistance (1, 17). 20-HETE possesses biological effects that can potentially contribute to these physiological changes during pregnancy. These biological effects include inhibition of ion transport along the nephron and vasoconstriction of renal arterioles (24, 33). We demonstrated distinct upregulation of CYP4A expression and 20-HETE synthesis in renal microvessels from rats on days 6 and 12 of gestation, which returned to control levels at day 19 of gestation (40). The factors responsible for the reduction of CYP4A expression and 20-HETE synthesis in late pregnancy are not known. We considered a role for nitric oxide (NO) in the regulation of CYP4A expression and activity in renal microvessels during late pregnancy because inhibition of NO synthesis increases 20-HETE synthesis (31), NO donors decrease the production of 20-HETE in renal microvessels (36), and NO production increases in pregnancy (1, 5).
The present study was undertaken to explore possible biochemical mechanisms underlying the effect of NO on CYP4A protein expression and activity and to examine whether inhibition on NO synthesis alters renal vascular 20-HETE synthesis in late pregnancy. We showed that NO readily binds to the heme moiety of the major CYP4A isoforms expressed in female rats, CYP4A1 and CYP4A3, with distinct isoform-specific affinity and that peroxynitrate increases tyrosine nitrosylation of these proteins. We also showed that inhibition of NO synthesis during the third week of gestation leads to a marked increase in vascular 20-HETE synthesis. Furthermore, coadministration of 1-aminobenzotriazole (ABT), a CYP4A inhibitor, prevents this increase. These changes in vascular 20-HETE synthesis were associated with reciprocal changes in systolic blood pressure, suggesting that alteration in vascular 20-HETE synthesis may contribute to the regulation of blood pressure during pregnancy.
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MATERIALS AND METHODS |
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Animals. All animals were purchased from Charles River Laboratories, Wilmington, MA. Experiments were conducted in male and female Sprague-Dawley rats (8 wk old), pregnant (timed pregnancy), and the same age control female Sprague-Dawley rats. Experimental protocols were approved by the Institutional Animal Care and Use Committee. Rats were maintained under controlled housing conditions of light and temperature and received standard laboratory chow and water until used.
Quantitative competitive RT-PCR for CYP4A2. The detailed procedure
for the preparation of competitor DNA of CYP4A2 was described in the
manufacturer's protocol (PanVera, Madison, WI). This method was designed to
generate CYP4A2 competitor (200 nucleotides) that is 117 nucleotides less than
the target CYP4A2 cDNA. We used two sets of PCR to generate CYP4A2 competitor
DNA. The first PCR primer set contained sequences that hybridize to a CYP4A2
sequence and are flanked by DNA-specific sequences according to the
manufacturer's protocol. The product of the first PCR set was purified and
used as the template for the second PCR set. The second PCR primer set
contained sequences that hybridize to a CYP4A2 sequence and are flanked by
SP6 promoter-specific sequences. The product of the second PCR set
was purified and used to generate CYP4A2 competitor RNA by in vitro
transcription using a SP6 RNA polymerase. The template was then
digested with DNase I, and the RNA was purified by phenol/chloroform/isoamyl
alcohol method. The amount of the competitor RNA synthesized was quantified by
spectrophotometry. Aliquots of total RNA (5 µg) from the kidneys of male
and female rats were prepared and a x5 dilution series of competitor RNA
(1,000; 200; 40; 8; 1.6; 0.32; 0.06 pg, respectively) was added into these
aliquots, and RT-PCR was performed. A reverse transcription reaction was
performed using a first-strand cDNA synthesis kit (Pharmacia Biotech,
Milwaukee, WI) as previously described
(38). After RT-PCR, aliquots
(10 µl) of PCR product were electrophoresed on a 2% agarose gel and
visualized by ethidium bromide staining. Gel pictures were scanned and
densitometry analysis was performed with Scion Image software using gray color
scale as a standard. The ratio of the density of the competitor RNA to the
CYP4A2 RNA, plotted against the amount of the competitor RNA added to each
reaction, was used to estimate CYP4A2 mRNA levels as described
(30). The sequences of the
primers used were as follows: CYP4A2 +
DNA: 5'-AGA TCC AAA GCC
TTA TCA ATC GTA CGG TCA TCA TCT GAC AC-3' (forward primer), 5'-CAG
CCT TGG TGT AGG ACC TTC ATT ACG CAT CGC TAT TAC-3' (backward primer);
and SP6 + CYP4A2: 5'-ATT TAG GTG ACA CTA TAG AAT ACA GAT CCA
AAG CCT TAT CAA TC-3' (forward primer), 5'-CAG CCT TGG TGT AGG ACC
TTC ATT ACG CAT CGC TAT TAC-3' (backward primer).
Preparation of recombinant CYP4A membranes. CYP4A proteins were expressed using the baculovirus-Sf9 insect cell expression system as described previously (28). CYP4A recombinant Sf9 cell membranes were prepared after infection with the recombinant virus and incubation in the presence of hemin (4 µg/ml) for 72 h followed by centrifugation at 100,000 g for 60 min of cell lysates as described (28). The membrane pellets were resuspended in sucrose buffer (50 mM potassium phosphate, pH 7.4, and 0.5 M sucrose) and stored at 80°C. Protein concentration was determined according to the method of Bradford (Bio-Rad, Melville, NY). CYP content was calculated from the reduced CO-difference spectrum using an extinction coefficient of 91 mM (28).
Effect of sodium nitroprusside and peroxynitrite on recombinant CYP4A catalytic activity. The stability of the NO donor sodium nitroprusside (SNP) was examined with NO-sensitive litmus paper using Griess reagent [0.5 g of sulfanilamide plus 20 mg of N-(1-naphthyl)ethylenediamine dihydrochloride] dissolved in 10 ml of methanol (29). In our preliminary study, SNP constantly released NO during a 10- to 20-min incubation (data not shown). Peroxynitrite (PN) stock was diluted in 0.3 M sodium hydroxide, and the concentration was determined by the extinction coefficient of 1,670 M · cm1 · 302 nm1 (3). SNP (0.011 mM) or PN (0.011 mM in 0.3 N NaOH) was added to mixture containing recombinant CYP4A1 or 4A3 membranes, purified NADPH-CYP oxidoreductase, and cytochrome b5 at a molar ratio of 1:14:4. This mixture was preincubated with NADPH (1 mM) in a final volume of 0.15 ml of buffer (10 mM MgCl2 and 100 mM KH2PO4, pH 7.2). The mixtures were preincubated at room temperature for 20 min. [1-14C]AA (0.4 µCi, 7 nmol) was then added, and incubation was carried out at 37°C for 30 min. Control incubations included the vehicle of SNP or PN. The reaction was terminated by acidification to pH 3.54.0 with 2 M formic acid, and metabolites were extracted with ethyl acetate. The final extract was evaporated under nitrogen, resuspended in 50 µl of methanol, and injected onto the HPLC column. Reverse-phase HPLC was performed on a 5-µm ODS-Hypersil column, 4.6 x 200 mm (Hewlett-Packard, Palo Alto, CA) using a linear gradient ranging from acetonitrile:water:acetic acid (50:50:0.1) to acetonitrile:acetic acid (100:0.1) at a flow rate of 1 ml/min for 30 min. The elution profile of the radioactive products was monitored by a flow detector (In/us System, Tampa, FL). The identity of 20-HETE was confirmed by its comigration with an authentic standard. Twenty-HETE formation was estimated based on the specific activity of the added [1-14C]AA and was expressed as nanomoles per minute per nanomoles of P-450.
Nitration of tyrosine residues of soluble CYP4A proteins by PN. Sf9 cell membranes containing recombinant CYP4A1 or 4A3 were suspended in 2 ml of ice-cold immunoprecipitation buffer {50 mM Tris · HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, protease inhibitor cocktail [aprotinin, leupeptin, pepstatin (1 mg/ml each)]}. The mixtures were incubated on an orbital shaker at 4°C for 15 min. Soluble CYP4A proteins were obtained by centrifuging at 14,000 g at 4°C for 15 min. CYP content was determined by using the reduced CO-difference spectrum. Soluble CYP4A1 or 4A3 proteins (25 pmol) were incubated with PN (1 mM) or vehicle control in a final volume of 1 ml immunoprecipitation buffer at room temperature for 30 min. The reaction mixtures were incubated with protein G-agarose/sepharose at 4°C for 10 min and spun down by centrifuge to reduce nonspecific binding. Anti-CYP4A antibody was added to the mixtures (10 µg antibody/25 pmol CYP4A). The CYP4A/antibody mixtures were incubated overnight at 4°C. The immunocomplex was captured with 100 µl of protein G-agarose/sepharose by gently rocking for 2 h at 4°C. The immunoprecipitation product was collected by pulse centrifugation (5 s at 14,000 rpm). The pellet was washed three times with PBS. The pellet was then resuspended with 60 µl of sample buffer and boiled for 5 min. The agarose/sepharose beads were collected by centrifugation, and SDS-PAGE was performed using the supernatant. Nitration of tyrosine residues of CYP4A proteins was determined by immunoblotting with anti-3-nitrotyrosine antibodies (Up-state Biotechnology).
Protocol to evaluate the effect of inhibition of NO synthase and NO synthase plus CYP4A on systolic arterial blood pressure, urinary NO2/NO3 excretion, and renal microvessal 20-HETE synthesis. Rats were placed in metabolic cages on the gestational day 13. On the gestational day 15, rats were treated with NG-nitro-L-arginine methyl ester (L-NAME; 0.25 mg/ml in drinking water) or L-NAME (0.25 mg/ml in drinking water) plus ABT (25 mg/kg ip) for 6 days (days 15 through 20 of pregnancy). The dosage of L-NAME treatment used for this study was based primarily on a literature search (15, 31). The dosage of ABT used was based on a previous study (40). Pregnant rats in the control group were treated with water. Systolic arterial blood pressure was measured daily by tail-cuff sphygmography using a Natsume KN-210 apparatus (Peninsula Laboratories, Belmont, CA). Rats were warmed at 40°C for 10 min and allowed to rest quietly in a Lucite chamber before tail-cuff sphygmographgy; 10 pressure measurements were recorded for each rat, and the average systolic blood pressure was calculated. Urinary NO2/NO3 excretion was determined by a fluorometric method (Cayman, MI). After treatment, the rats were killed on day 21 of gestation and kidneys were removed for the preparation of microvessels to measure 20-HETE synthesis.
Measurement of 20-HETE synthesis in microvessels. Renal microvessels were isolated by microdissection and homogenates of tissue were prepared as described previously (23). Homogenates of microvessels (30 µg protein) were incubated with AA (20 µM) in 1 ml of assay buffer containing 100 mM potassium buffer (pH 7.4), 10 mM MgCl2, 1 mM NADPH, and 2 µM indomethacin for 60 min at 37°C. After incubation, [20,20-2H2]20-HETE (1 ng) was added as an internal standard, and the reaction mixture was acidified to pH 4 with 1 M formic acid. The mixture was extracted twice with 2 ml of ethyl acetate. The final extract was subjected to reverse-phase HPLC. Fractions coeluting with the 20-HETE standard were collected, evaporated to dryness, and derivitized to the pentafluorobenzyl bromide ester trimethylsilyl ether. 20-HETE was quantitated by negative chemical ionization-gas chromatography/mass spectrometry (NCI-GC/MS) by comparing the ratio of ion intensity (391:393) for derivatized 20-HETE vs. derivatized [20,20-2H2]20-HETE (39).
Statistical analysis. Data are expressed as means ± SE. All data were analyzed by a one-way analysis of variance or the Student's t-test for unpaired samples. Statistical significance was set at P < 0.05.
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RESULTS |
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Interaction between NO donor and baculovirus-expressed CYP4A isoforms
in vitro. To examine whether NO binds to the major CYP4A proteins
expressed in female rats, recombinant CYP4A1 and CYP4A3 were incubated with
the NO donor SNP (1 mM) at room temperature for 20 min. A representative
visible light absorption spectrum was shown in
Fig. 2A. Incubation of
CYP4A3 membranes with SNP increased absorption at 440 nm, indicating the
formation of ferric-nitrosyl complexes at the CYP-heme binding site
(36). More interestingly, the
absorbance at 440455 for CYP4A3 was about twofold
higher than that of CYP4A1 (optical density of 0.01 for CYP4A3 vs. 0.0045 for
CYP4A1). In other words, the binding affinity of NO to the heme moiety of
CYP4A3 was about twofold stronger than for CYP4A1. These results reveal a
significant difference in the binding characteristic of NO to the heme moiety
of these two isoforms. Moreover, addition of SNP (0.011 mM) inhibited
both CYP4A1- and CYP4A3-catalyzed AA
-hydroxylation in a
concentration-dependent manner. At low concentrations, SNP had a greater
inhibitory effect on CYP4A3-catalyzed activity than on CYP4A1
(Fig. 2B). Taken
together, these results suggest that the greater inhibitory effect of SNP on
CYP4A3 may be due to greater binding affinity of NO for the heme moiety of
CYP4A3.
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Effect of PN on recombinant CYP4A proteins. Incubation of PN (0.011 mM) with recombinant CYP4A1 and CYP4A3 membranes caused a concentration-dependent inhibition of both CYP4A1- and CYP4A3-catalyzed 20-HETE synthesis. PN had a greater inhibitory effect on CYP4A3-catalyzed activity than CYP4A1 (Fig. 3A). To examine whether PN can modify tyrosine residues of CYP4A isoforms, soluble preparations of recombinant CYP4A1 and CYP4A3 were incubated with 1 mM PN at room temperature for 30 min. CYP4A proteins were then isolated from the reaction mixtures by immunoprecipitation with CYP4A-specific antibody, and the nitration of tyrosine residues of CYP4A proteins was determined by Western blot analysis with anti-3-nitrotyrosine antibody. As shown in Fig. 3B, a strong 3-nitrotyrosine-immunoreactive band was observed when CYP4A proteins were incubated with PN. These results suggest that the nitration of tyrosine residues of CYP4A proteins by PN may contribute to PN inhibitory action on CYP4A-catalyzed 20-HETE formation.
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Effect of SNP and PN on renal microvessel 20-HETE synthesis. To examine whether NO and PN have a similar effect on 20-HETE synthesis in renal microvessels isolated from female rats, homogenates were preincubated with SNP (1 mM), PN (1 mM), or vehicle control at room temperature for 30 min followed by incubation with AA and NADPH. 20-HETE synthesis was determined by NCI-GC/MS. As shown in Fig. 4, SNP and PN caused 59 and 65% inhibition of renal microvessel 20-HETE synthesis, suggesting that NO and PN act as negative regulators of 20-HETE synthesis in renal microvessels.
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Effect of NO synthase and NO synthase/CYP4A inhibition on systolic blood pressure, urinary NO2/NO3 excretion, and renal microvessel 20-HETE synthesis. L-NAME (0.25 mg/ml in drinking water), L-NAME (0.25 mg/ml in drinking water) plus ABT (25 mg/kg ip), or vehicle control was administered for 6 days to pregnant rats beginning on day 15 of gestation. As seen in Table 1, systolic blood pressure in L-NAME-treated rats was significantly increased compared with pregnant control rats, whereas systolic blood pressure in L-NAME plus ABT-treated group remained unaffected. Urinary NO2/NO3 excretion, an index for whole body production of NO in pregnant rats and women (5, 6), decreased by 40% (P < 0.05) following L-NAME treatment (Table 1). Interestingly, renal microvessel 20-HETE synthesis increased threefold relative to control in the L-NAME-treated group but was unchanged in the L-NAME plus ABT-treated group (Table 1).
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DISCUSSION |
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During pregnancy in rats, urinary excretion and plasma levels of nitrate are elevated and urinary excretion of cGMP is also increased. Chronic treatment with L-NAME reduces urinary nitrate excretion in pregnant rats (5). Moreover, the binding of NO to hemoglobin is only detected in the blood of pregnant, but not nonpregnant, rats (5). Augmentation of NO production and increased expression levels of renal neuronal NO synthase (NOS) and inducible NOS have been observed during normal pregnancy (1), and chronic inhibition of NO synthesis by L-NAME during pregnancy resulted in preeclampsia-like symptoms (26, 41). These reports provided substantial evidence to implicate NO as a major contributor to the implementation of a vasodilatory state in pregnancy (18, 35). On the basis of this information and on data indicating that NO inhibits CYP4A expression and 20-HETE synthesis and action (31, 36), we postulated the existence of interactions between NO and CYP4A/20-HETE that may explain the decreased renal vascular 20-HETE synthesis during the third week of pregnancy and may contribute to the regulation of vascular tone and blood pressure during pregnancy. In the present study, we showed that NO binds to the heme moiety of CYP4A1 and CYP4A3, the major isoforms expressed in female rats, and inhibits their catalytic activity. We also demonstrated that PN, which shows elevated production of NO and superoxide (7, 22), causes nitrosylation of tyrosine residues on CYP4A1 and CYP4A3.
The CYP4A enzymes (CYP4A1, 4A2, 4A3, and 4A8) are considered to be the
major AA -hydroxylases in the rat kidney and thereby the primary
contributors of 20-HETE synthesis. With the use of quantitative RT/PCR and
Western blot analysis, we demonstrated that CYP4A1 and CYP4A3 are the major
CYP4A expressed in the kidneys of female rats. The expression levels of CYP4A2
measured by quantitative RT/PCR were 18-fold lower in female rats compared
with male rats. These results are in accordance with other reports documenting
lower and even undetectable expression levels of CYP4A2 in the kidneys of
female rats (37) as well as
reports demonstrating androgen-dependent expression of CYP4A2
(11). As for the CYP4A8
expression, a recent study by Holla et al.
(10) suggested that CYP4a12 (a
murine homologue gene to CYP4A8) is a male-specific and androgen-regulated
enzyme and has very low expression in female kidneys. Nakagawa et al.
(27) showed that CYP4A8
expression in the rat is androgen sensitive. CYP4A8 protein has a similar
electrophoretic mobility as CYP4A2
(28); the absence of
CYP4A2-immunoreactive protein in renal microsomes from female rats
(Fig. 1) suggests low
expression levels of CYP4A8. On the basis of these reports, our data, and
previous studies showing that the recombinant CYP4A1 and CYP4A3 proteins
catalyze AA
-hydroxylation to 20-HETE, it is likely that these isoforms
contribute significantly to renal 20-HETE synthesis in female rats. However,
we cannot rule out the possibility that other isoforms of the CYP4 gene family
such as CYP4F proteins, CYP4F1, CYP4F4, CYP4F5, and CYP4F6, may be involved in
the renal production of 20-HETE in female rats
(4,
16). Kalsotra et al.
(14) showed significant levels
of expression of all CYP4F isoforms in kidneys of female rats and further
documented an estrogen-sensitive expression of CYP4F1, CYP4F4, and CYP4F6. The
catalytic activity of CYP4F isoforms toward AA and their ability to catalyze
the production of 20-HETE has not been fully examined. Further studies that
allow evaluation of the distinct contribution of each of the CYP4A and 4F
isoforms to 20-HETE synthesis are needed.
NO inhibits heme-containing proteins such as CYP (25). The mechanisms of the interaction between NO and CYP proteins were described by Minamiyama et al. (25), who demonstrated that NO can interact with CYP in two ways: NO binds reversibly with the heme moiety and irreversibly with cysteine residues of CYP proteins. These NO-CYP adducts are enzymatically inactive in vitro. Moreover, Roberts et al. (32) demonstrated that PN can modify tyrosine residues of CYP2B1 and inactivate CYP2B1-catalyzed reaction. It is likely that NO interacts with the major CYP4A isoforms in female rats, i.e., CYP4A1 and 4A3, in a similar manner. However, it is difficult to study the interaction between NO and CYP4A isoforms in renal tissues because renal tissues contain numerous CYP enzymes other than CYP4A isoforms. Baculovirus-expressed CYP4A isoforms provide a unique tool to study the interaction between NO and individual CYP4A isoforms in vitro because there is a negligible level of CYP content in Sf9 insect cells (39). Our results indicated that NO binds to the heme moiety of CYP4A1 and CYP4A3 with different affinities. The heme moiety of CYP enzymes is essential for the oxidation reaction. NO binding to the heme moiety can interfere with the electron transport mechanisms of the oxidation reaction. It is possible that the ability of NO to inhibit CYP4A3-catalyzed 20-HETE synthesis to a greater extent than that of CYP4A1 is due to the higher binding affinity of NO to CYP4A3. PN, a powerful oxidant, is derived from NO and superoxide. Because CYP isoforms can generate varying amounts of oxygen-derived free radicals such as superoxide ion during the catalytic cycle of CYP enzymes (8), it is possible that superoxide ion generated from the CYP4A-catalyzed reaction can interact with NO and cause the formation of PN. We showed that PN inhibits 20-HETE synthesis catalyzed by CYP4A1 and CYP4A3. The mechanisms underlying this inhibition are not clear; however, the ability of PN to nitrosylate tyrosine residues of CYP4A1 and CYP4A3 may constitute, at least in part, a mechanism of inhibition. However, additional studies are needed to demonstrate that tyrosine nitrosylation of CYP4A proteins occurs in vivo and that nitrosylated CYP4A proteins are catalytically inactive.
In contrast to normal pregnancy, preeclampsia is characterized by increased arterial blood pressure, generalized vasoconstriction, increased systemic resistance, widespread vascular endothelial damage, decreased fetal growth, and proteinuria (21). The exact mechanisms that mediate preeclampsia are still unknown. Several reports have suggested that NO may play an important role in its development (2, 22). Moreover, two reports demonstrated that chronic inhibition of NO synthesis in late pregnancy in rats resulted in signs similar to those of preeclampsia (26, 31). That the increased renal microvessel production of 20-HETE following administration of L-NAME during the third week of pregnancy together with reports that NO inhibits 20-HETE synthesis and interferes with 20-HETE vasoconstrictor activity in vivo (31, 36) suggest the contribution of 20-HETE to the implementation of renal vasoconstriction and increased blood pressure (Table 1) under conditions where NO production is suppressed. This notion is further substantiated by data showing that coadministration of ABT, an inhibitor of CYP4A activity, abolished the L-NAME-induced increase in blood pressure in these rats (Table 1).
In summary, this study provides the first evidence to show that NO binds differently to the heme of CYP4A isoforms and inhibits 20-HETE synthesis by recombinant CYP4A proteins and renal microvessels in female rats. This study also shows that PN modifies tyrosine residues of CYP4A proteins and inhibits their catalytic activity. Additional data show that augmentation of renal microvessel 20-HETE synthesis after NOS inhibition is associated with increased blood pressure and that this increase is negated by treatment with a CYP4A inhibitor. Hence, this study offers evidence that NO acts as a buffer system to counteract 20-HETE-mediated vasoconstriction mechanisms during pregnancy.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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