Cytotoxicity by nitrite inhalants is not related to peroxynitrite formation

Toxicology Letters 132 (2002) 37 – 45 www.elsevier.com/locate/toxlet

Cytotoxicity by nitrite inhalants is not related to peroxynitrite formation Lee S.F. Soderberg *, Usha Ponnappan Department of Microbiology and Immunology, Uni6ersity of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205, USA Received 5 November 2001; received in revised form 19 February 2002; accepted 20 February 2002

Abstract Nitrite inhalant abuse has been correlated with HIV and Kaposi’s sarcoma. Mouse models of inhalant exposure show immunosuppression and loss of immune cells. In the present study, isobutyl nitrite caused a dose-dependent loss of viability of a macrophage cell line. In the absence of cells, isobutyl nitrite reacted with hydrogen peroxide to form peroxynitrite. However, assays of mitochondrial respiration and nitration that detect peroxynitrite indicated that very little was present in cell cultures following exposure to the inhalants. Isobutyl, isoamyl, and butyl nitrites inhibited mitochondrial respiration, but only at high concentrations. Similarly, the nitrating activity of isobutyl nitrite occurred only at high concentrations and was not affected by the presence of hydrogen peroxide. Western blots showed that the inhalant did not increase nitrotyrosine formation in RAW cells or in peritoneal exudate macrophages (PEM) from exposed mice. Thus, the toxicity induced by isobutyl nitrite was probably not due to peroxynitrite formation. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Inhalant; Peroxynitrite; Macrophage; Kaposi’s sarcoma; AIDS

1. Introduction The alkyl nitrites, including isobutyl, amyl, butyl, and cyclohexyl nitrites, are small, volatile compounds that are abused by inhalation. Epidemiological studies have identified nitrite inhalant abuse as an independent risk factor for seropositivity to HIV (Moss et al., 1987; Chesney

* Corresponding author. Tel.: +1-501-686-6368; fax: + 1501-686-5359. E-mail address: [email protected] (L.S.F. Soderberg).

et al., 1998), HHV-8 (Pauk et al., 2000), and to the incidence of Kaposi’s sarcoma in AIDS patients (Marmor et al., 1982; Armenian, et al., 1993). If nitrite inhalant abuse impaired cellular immunity, it could lower resistance to viral infection and tumor growth. To study the immunotoxicity of these inhalants, we developed a mouse model of inhalant exposure that approximates the overall dose to which abusers are exposed. Mice were exposed in an inhalation chamber to 900 ppm isobutyl nitrite for 45 min/day for 14 days. Such an exposure compromised cytotoxic T cell induction (Soderberg, 1994), macrophage tumoricidal activity (Soderberg and Barnett, 1995), and

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promoted a 4-fold increase in the growth of syngeneic tumors (Soderberg, 1999). The losses in immune function were accompanied by a significant loss of immune cells. However, the cell loss alone could not account for the reduction in immune function. Even a single 45 min exposure of mice to the inhalant, which was not sufficient to impair T cell or macrophage cytotoxic activity, significantly reduced spleen cellularity by 39% (Guo et al., 2000). The loss of cells was apparently nonspecific, since the proportions of CD4+ and CD8+ T cells or B cells were unchanged in the spleen following inhalant exposure. It is likely that the reduction in cellularity was not due to cell mobilization into the circulation or to extravascular spaces, because exposure to the inhalant also reduced the numbers of peripheral blood leukocytes by 21% and the number of resident peritoneal macrophages by 21%. Inhalation of the volatile alkyl nitrites induces the relaxation of smooth muscles, vasodilation, increases heart rate, and decreases systolic blood pressure (Hadjimiltiades et al., 1991). As with the other nitrites, isobutyl nitrite has a very short half-life in vivo, rapidly degrading to isobutyl alcohol, which is much less toxic (Kielbasa and Fung, 2000b). The physiological effects of the inhalants have been attributed to the generation of nitric oxide (NO), either by enzymatic activity (Kowaluk and Fung, 1991) or by spontaneous release (Soderberg et al., 2000). However, concentrations of NO (115 ppm) comparable to the amount produced by immunotoxic doses of isobutyl nitrite did not reduce peripheral blood leukocyte counts or macrophage tumoricidal activity (Soderberg et al., 2000). Thus, it is not likely that the toxicity associated with the nitrite inhalants is solely due to NO generation. However, NO readily reacts with superoxide to form a much more toxic radical, peroxynitrite (Stamler, 1994). In addition, it has been reported that another nitrite compound, isoamyl nitrite, directly reacted with hydrogen peroxide to form peroxynitrite (Uppu and Pryor, 1996). In the present study, the toxicity of isobutyl nitrite was further explored. It was shown that, like isoamyl nitrite, isobutyl nitrite efficiently formed peroxynitrite under certain conditions, but apparently did not form signifi-

cant amounts of peroxynitrite in cell culture or in vivo.

2. Materials and methods

2.1. Cells and exposure The murine macrophage cell line, RAW 267.4, was maintained in RPMI 1640 medium, supplemented with 10% fetal calf serum, 50 nM 2-mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml streptomycin. The presence of 2-mercaptoethanol in the cell culture medium did not alter the results reported. Isobutyl, butyl, and isoamyl nitrites (Aldrich Chemical Co.) were stored at 4 °C under nitrogen. For addition to cell culture, the alkyl nitrites were mixed with equimolar amounts of ethanol to make them more soluble in water before dilution in RPMI 1640 medium. The diluted nitrites were added to triplicate cultures of RAW cells (2.5× 106 per culture). Following 45 min incubation at 37 °C, the cells were washed three times in medium before analysis.

2.2. Mouse exposure and macrophage preparation Isobutyl nitrite was vaporized in a flask and quantified with a halothane monitor (PuritanBennet, Model 222, Datex, Tewkesbury, MA) calibrated for isobutyl nitrite. Groups of five 6–8 week-old, female C57BL/6 mice were exposed to air or to 900 ppm isobutyl nitrite in an inhalation chamber for 45 min/day for 5 days. Macrophages were harvested by peritoneal lavage. Peritoneal exudate macrophages (PEM) were collected 4 days after intraperitoneal injection with 1 ml 3% thioglycollate. Macrophages were collected by lavage and cultured at 5× 105 cells per well in 96-well plates. Cells were maintained in RPMI 1640 medium, supplemented with 10% fetal calf serum, 50 nM 2-mercaptoethanol, 100 U/ml penicillin, and 100 mg/ml streptomycin. After 1 h incubation, nonadherent cells were removed by washing. Macrophages were incubated overnight in medium or medium containing 100 U/ml recombinant IFN-g (Genzyme) and 1 mg/ml LPS (Escherichia coli 05:B5, Sigma Chem.).

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2.3. Synthesis of peroxynitrite Peroxynitrite was prepared as described (Uppu and Pryor, 1996). Briefly, 30% hydrogen peroxide (2.8 ml) was diluted with 7 ml distilled, deionized water, 5 ml 0.04 diethylenetriaminepentaacetic acid (DTPA, Sigma) in 0.5 NaOH, and mixed gently on ice. The pH of the buffered H2O2 was adjusted to 12.5 by adding 7.5 ml 5 N NaOH. An equimolar amount of isoamyl or isobutyl nitrite (2.8 ml) was then added and the mixture was vigorously stirred at room temperature for 3.5 h. Using a separatory funnel, the aqueous phase was collected and washed twice with an equal volume of chloroform to remove isobutyl nitrite and isobutyl alcohol. Residual H2O2 was removed by passing the aqueous phase over a manganese oxide column, which had been washed with distilled water and 0.1 N NaOH. The aqueous phase was assayed for peroxynitrite by 2%-2%-azino-bis (3ethylbenz-thiazline 6-sulfonic acid ammonium salt) (ABTS, Sigma). ABTS (0.3 mg/ml in 0.1 M anhydrous citric acid adjusted to pH 4.35) was combined with DTPA (0.1 mM) and diluted 1:10. This mixture was added to dilutions of the aqueous phase and, after 10 min, absorbence was measured at 405 nm (m = 36 800 M − 1 cm − 1). Peroxynitrite was stored at −20 °C.

2.4. Respiration RAW 267.4 cells were incubated with a range of concentrations of isobutyl, butyl, isoamyl nitrite, or peroxynitrite for 45 min. Mitochondria were prepared by cell lysis in a homogenizer in phosphate buffer with 0.3 M sucrose, 5 mM 3(morpholino) propanesulfonic acid (Sigma), 5 mM K2PO4, 1 mM EGTA, and 0.1% bovine serum albumin (Cassina and Radi, 1996). Large cell debris was removed by centrifugation at 1500×g and mitochondria pelleted at 10 000×g and resuspended to 25–35 mg protein per ml and maintained at 4 °C until testing. Respiration was measured as the mitochondrial-dependent reduction of [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (MTT, Sigma) as described (Szabo´ et al., 1996). Briefly, mitochondrial preparations were incubated in RPMI 1640

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with 10% fetal calf serum and 0.2 mg/ml MTT (Sigma Chem. Co.). After 90 min, the medium was removed and the cells solubilized in 100 ml dimethyl sulfoxide. The extent of reduction of MTT to formazan was measured at 550 nm in a spectrophotometer. Data were reported as the mean percent of control respiration of triplicate cultures 9 standard error of two separate experiments.

2.5. Nitration acti6ity Isobutyl nitrite was assayed for the nitration of 4-hydroxyphenylacetic acid (4-HPA) (Uppu and Pryor, 1996). Dilutions of isobutyl nitrite and of peroxynitrite were reacted with 2 mM 4-HPA in 2 ml of 0.1 M phosphate buffer, pH 7.0, containing 0.1 mM DTPA (Sigma) at room temperature. After 5 min, the reaction was halted with 0.5 ml 0.5 N NaOH and nitration measured at 430 nm (m= 4400 M−1 cm−1) using a spectrophotometer.

2.6. Western blots Cytosolic extracts for Western blotting were prepared by homogenization of cells in lysis buffer (1 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM sodium orthovanadate, and 0.5% NP-40). Protease inhibitors were added prior to use (Trebilcock and Ponnappan, 1996). Cell lysates equalized for protein (40 mg) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to nitrocellulose, and immuno-blotted with antibody specific to nitrotyrosine (Upstate Biotech), and detected using anti-IgG coupled to horseradish-peroxidase (Transduction Labs) followed by Enhanced Chemiluminescence (ECL) (Amersham). Results are representative of at least two separate experiments.

3. Results Since even a single inhalation exposure of mice to inhaled isobutyl nitrite (900 ppm) resulted in reductions in spleen, peripheral blood, and peritoneal cell numbers (Guo et al., 2000), we wanted

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to determine if the inhalant was directly toxic to macrophages at relevant concentrations. A macrophage cell line, RAW 267.4 cells, was treated for 45 min with various concentrations of isobutyl, isoamyl, or butyl nitrite. After the 45 min incubation period, the cells were washed and stained with trypan blue and viable cells were counted. Immediately after inhalant exposure the number of viable RAW cells was reduced by an average of 19% at 1.5 mM (ANOVA, P B 0.001), 26% at 3 mM (PB 0.001), and 65% at 6 mM (P B0.001) (Fig. 1A). Isoamyl nitrite was somewhat more toxic at 6 mM than the other compounds (PB0.05). In addition, RAW cells were exposed for 45 min to various concentrations of isobutyl nitrite, washed, and the number of viable cells determined at intervals of incubation thereafter. At 6 mM isobutyl nitrite, viable cell numbers dropped by 60% (P B 0.01) immediately following the 45 min exposure (Fig. 1B) and continued to drop over the next hour (P B 0.05). Exposure of the cell cultures to 1.5 or 3 mM isobutyl nitrite caused an immediate drop of 30% (PB0.05) in viable cell numbers, however, cell counts did not significantly decline further upon incubation. We (Soderberg et al., 2000) and others

(Kowaluk and Fung, 1991) have shown that isobutyl nitrite produced NO, which was not as toxic as the inhalant. However, the much more toxic radical, peroxynitrite, forms when NO reacts with superoxide (Stamler, 1994). Peroxynitrite has also been shown to result from the reaction of isoamyl nitrite with hydrogen peroxide (Uppu and Pryor, 1996). To determine if isobutyl nitrite similarly forms peroxynitrite, isobutyl nitrite or isoamyl nitrite was mixed under alkaline conditions for 3.5 h with equimolar concentrations of H2O2. Excess H2O2 and nitrite compounds were removed, as described (Uppu and Pryor, 1996). Assays for peroxynitrite showed that isobutyl nitrite reacted with H2O2 to form peroxynitrite with an efficiency similar to that of isoamyl nitrite (Fig. 2). The molar ratio of peroxynitrite formation from isobutyl nitrite of 0.185% was only slightly different than from isoamyl nitrite, 0.166%. Thus, under controlled, alkaline conditions the inhalants readily generated peroxynitrite when reactive oxygen species (ROS) were present. It is possible that ROS generated by macrophages could react with isobutyl nitrite or with NO produced by isobutyl nitrite to form toxic peroxynitrite. However, the peroxynitrite assay is not sensitive enough to detect peroxynitrite at physiological levels.

Fig. 1. In vitro cytotoxicity of nitrite inhalants. RAW cells (2.5 × 106/ml) were exposed for 45 min to 0, 1.5, 3, or 6 mM isobutyl, isoamyl, or butyl nitrite (A). Viable cells were counted using trypan blue. Alternatively, cells were treated for 45 min with 0, 1.5, 3, or 6 mM isobutyl nitrite, washed, and viable cells counted immediately or after 0.5, 1, or 4 h (B). Data, reported as mean viable cells per culture 9 standard error of triplicate samples, are representative of two separate experiments.

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Fig. 2. Isobutyl nitrite reacted with H2O2 to form peroxynitrite. Isobutyl or isoamyl nitrites were mixed for 3.5 h with equimolar concentrations of H2O2. Excess nitrite and H2O2 were removed and peroxynitrite was measured by the ABTS assay. The data are representative of six separate experiments.

A sensitive indication of peroxynitrite formation at the cellular level is inhibition of mitochondrial respiration (Szabo´ et al., 1998). To test this with the inhalants, RAW cells were treated for 45 min with various concentrations of peroxynitrite or isobutyl, isoamyl, or butyl nitrite. After washing the cells, mitochondrial respiration was assayed. Peroxynitrite efficiently inhibited mitochondrial respiration of the RAW cells with a 50% inhibitory concentration (IC50) of 350 mM (Fig. 3). Incubation of the macrophages with isobutyl nitrite also inhibited mitochondrial respiration, but only at concentrations above 25 mM with an IC50 of 100 mM. Thus, isobutyl nitrite was 285-fold less inhibitory of respiration than peroxynitrite (ANOVA, P B 0.001). Isoamyl nitrite had an inhibitory activity comparable to that of isobutyl nitrite, while butyl nitrite was only inhibitory at 200 mM. This suggests that, if the inhalants formed peroxynitrite in cell culture, the levels were too low to affect cell viability. Furthermore, even considering the differences in assays, these inhalant concentrations were too high to account for the loss of viability of the RAW cells. It is likely that the inhalants induced cell death by mechanisms other than inhibition of respiration. Another indicator of peroxynitrite formation is the nitration of a variety of cellular proteins,

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Fig. 3. The nitrite inhalants inhibited cellular respiration only at higher concentrations. Various concentrations of isobutyl, isoamyl, or butyl nitrite or peroxynitrite were added to RAW cell cultures. After 45 min, mitochondrial preparations were assayed for respiration by the MTT assay. Data are reported as mean percent of control respiration 9 standard error of triplicate samples for two separate experiments.

particularly at tyrosine residues (Beckman et al., 1996). To determine the relative nitrating activity of the inhalants, we measured the nitration of 4-HPA with peroxynitrite and isobutyl nitrite. As shown in Fig. 4, peroxynitrite efficiently nitrated

Fig. 4. Nitration by isobutyl nitrite was not affected by the presence of H2O2. Dilutions of isobutyl nitrite and of peroxynitrite were reacted with 4-HPA for 5 min at room temperature. Nitration was measured at 430 nm using a spectrophotometer. A constant 3 mM isobutyl nitrite was also assayed for nitration in the presence of various concentrations of H2O2. Data are reported as mM nitro-4-HPA formed and are representative of three separate experiments.

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4-HPA at a half-maximal concentration of 700 mM. Thus, 4-HPA nitration was slightly less sensitive than mitochondrial respiration to the presence of peroxynitrite. Isobutyl nitrite also nitrated 4-HPA, but at 30-fold higher concentrations. Interestingly, isobutyl nitrite was 5-fold more efficient at nitration than it was in the inhibition of respiration. Since isobutyl nitrite might form peroxynitrite more readily in the presence of ROS, we tested nitrating activity of a constant level (3 mM) of isobutyl nitrite mixed with varying concentrations of H2O2. The addition, of H2O2 did not significantly alter the nitrating activity of isobutyl nitrite, suggesting that the presence of ROS did not increase peroxynitrite formation under standard culture conditions. Another indicator of peroxynitrite formation is tyrosine nitration. To determine if isobutyl nitrite acted on cells to form nitrotyrosine, RAW cells were treated for 45 min in culture with relatively high concentrations of peroxynitrite (30 mM) or isobutyl nitrite (6 mM). Cell lysates were prepared immediately, standardized for protein levels, and analyzed for nitrotyrosine formation by Western blot. As shown in Fig. 5, peroxynitrite increased tyrosine nitration, particularly a major band at about 65 kDa. Several larger proteins also showed increased nitrotyrosine formation due to peroxynitrite exposure. In contrast, isobutyl nitrite actually reduced nitrotyrosine formation below the levels in control cells. Thus, not only did isobutyl nitrite not induce tyrosine nitration, the exposure apparently interfered with normal nitrotyrosine formation. Since isobutyl nitrite might act differently in vivo, we looked for protein nitration following inhalation exposure of mice. Groups of five mice were exposed to 0 or 900 ppm isobutyl nitrite for 45 min per day for 5 days, an exposure regimen previously shown to impair macrophage tumoricidal activity (Soderberg and Barnett, 1995). PEMs were collected and incubated in medium or activated with IFNg and LPS. Cell lysates were prepared 4 h later and were assayed for nitrotyrosine formation by Western blot. As shown in Fig. 6, many proteins stained with antinitrotyrosine antibodies (Upstate Biotech). Only slight differences in intensity were observed. Densitometer scanning of the four strongest bands

Fig. 5. RAW cell nitrotyrosine formation. RAW cell cultures were incubated with 30 mM peroxynitrite or 6 mM isobutyl nitrite. Cell lysates were prepared immediately, standardized for protein levels, resolved by SDS-PAGE, and analyzed for nitrotyrosine formation by Western blot. The data are representative of two separate experiments.

heavier than 25 kDa (43, 40, 34, 30 kDa) identified some minor changes. In these proteins, activation of macrophages from control mice consistently increased (12–43%) nitrotyrosine formation. With the exception of one of the bands (43 kDa) that was unchanged, the same bands from unstimulated macrophages derived from inhalant exposed mice showed reduced (− 15 to 29%) nitrotyrosine staining, compared with unstimulated control cells. Interestingly, this reflected the reduced nitrotyrosine formation of RAW cells treated with isobutyl nitrite (Fig. 5). Activation of macrophages from nitrite exposed mice did not induce consistent changes. Nitrotyrosine formation was increased in two bands (40 kDa 32%; 30 kDa 12%), although to levels that were still lower than stimulated control cells. Nitrotyrosine formation was further decreased in one band (43 kDa 19%) and in one band (34 kDa) it was not changed following activation of in-

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halant exposed cells. Thus, exposure to the inhalant did not show expected increases in nitrotyrosine formation and, in fact, appeared to slightly reduce nitrotyrosine levels in at least some proteins.

4. Discussion We have reported that mice exposed to abusive doses of isobutyl nitrite experienced dramatic losses of spleen, peripheral blood and peritoneal cavity cellularity (Guo et al., 2000). The present study suggests that the inhalant could directly kill cells in culture. Significant loss of viability occurred at only 1.5 mM isobutyl nitrite. Abusers expose themselves only for short intervals, but to much higher levels of isobutyl nitrite, estimated to be about 7000 ppm (Soderberg et al., 1996b). Perhaps the most direct comparison of human

Fig. 6. Inhalation exposure to isobutyl nitrite did not increase nitrotyrosine formation in macrophages. Groups of 5 mice were exposed to 0 or 900 ppm isobutyl nitrite for 45 min/day for 5 days. PEM were collected and activated with IFNg and LPS. Cell lysates were prepared, standardized for protein levels, resolved by SDS-PAGE, and analyzed for nitrotyrosine formation by Western blot. Arrows indicate major bands measured by densitometer. The data are representative of five separate experiments.

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and mouse exposure levels is methemoglobin formation. One of the major effects of exposure to the inhalants due to the oxidization of hemoglobin, methemoglobin formation has been reported to be as high as 39% in abusers (Goulle et al., 1994). Methemoglobinemia levels in our mouse model were 20% (Soderberg et al., 1996a), reinforcing our estimates that mouse exposure levels were comparable to and possibly lower than human exposures. Furthermore, the loss of cell viability following exposure of cells in culture to 3 mM isobutyl nitrite (− 26%) was comparable to in vivo losses in cellularity in the mouse model. Since isoamyl nitrite reacts with hydrogen peroxide to form the highly toxic radical, peroxynitrite (Uppu and Pryor, 1996), it is possible that inhaled isobutyl nitrite could similarly react with ROS produced by macrophages to form toxic peroxynitrite. Isobutyl nitrite did react with hydrogen peroxide to form peroxynitrite and the efficiency of the inhalant in forming peroxynitrite was comparable to that of isoamyl nitrite. However, the alkaline conditions necessary for peroxynitrite formation were not physiological. Moreover, functional assays of respiration and nitrating activity showed that isobutyl nitrite had much lower activity than peroxynitrite. The inhalant was nearly 300-fold less toxic to mitochondrial respiration than peroxynitrite and had 30-fold less nitrating activity. The presence of exogenous hydrogen peroxide in cell culture did not increase the nitrating activity of isobutyl nitrite. These characteristic activities have been used to detect peroxynitrite formation (Radi, 1998). Thus, if peroxynitrite was formed by the inhalant in cell culture, the levels were very low. These assays also suggest that the loss in viability due to isobutyl nitrite was not due to inhibition of respiration and probably not due to nitrating activity. If anything, exposure to the inhalant reduced nitrotyrosine formation. This was shown in RAW cells and marginally in macrophages from mice exposed to the inhalant. The reduction in nitrotyrosine formation following exposure to the inhalant was consistent with reduced inducible NO generated by macrophages from mice exposed to the inhalant (Soderberg and Barnett, 1995). Endogenous NO has been shown to react with a

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variety of proteins, including mitochondrial proteins involved in respiration, forming nitrotyrosine (Aulak et al., 2001). Although not thoroughly characterized, tyrosine nitration is known to alter at least some protein functions (Stamler, 1994). It has been reported that exposure to isobutyl nitrite increased tyrosine nitration in liver and kidney cells from rats exposed to the inhalant (Kielbasa and Fung, 2000a). We were unable to confirm that with macrophages from mice exposed to the inhalant. While the inhalants did not increase tyrosine nitration, they might act on other targets, including cysteine methionine, and tryptophan residues, sulfhydral groups, lipids, and iron – sulfur centers.

Acknowledgements The authors would like to express their appreciation to Jianhui Du for technical assistance. This work was supported by USPHS grant DA06662.

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