Identification of a Rat Liver Microsomal Esterase as a Target Protein

To identify proteins targeted by bromobenzene metabolites, we incubated [14C]bromobenzene in vitro with liver microsomes from phenobarbital-induced ra...
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Chem. Res. Toxicol. 1998, 11, 178-184

Identification of a Rat Liver Microsomal Esterase as a Target Protein for Bromobenzene Metabolites Elizabeth M. Rombach and Robert P. Hanzlik* Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045-2506 Received May 8, 1997

The hepatotoxicity of bromobenzene and many other simple organic molecules has been associated with their biotransformation to chemically reactive metabolites and the subsequent covalent binding of those metabolites to cellular macromolecules. To identify proteins targeted by bromobenzene metabolites, we incubated [14C]bromobenzene in vitro with liver microsomes from phenobarbital-induced rats under conditions which typically led to covalent binding of 2-4 nmol equiv of bromobenzene/mg of protein. Microsomal proteins were solubilized with detergent, separated by chromatography and electrophoresis, and analyzed for 14C by phosphorimaging of stained blots. Much of the radioactivity was associated with several bands of proteins of ca. 50-60 kDa, plus another prominent band around 70 kDa, but labeling density appeared to vary considerably overall. A major radiolabeled protein was purified by preparative electrophoresis and submitted to automated Edman microsequencing. Its N-terminal sequence was found to correspond to that of a known rat liver microsomal carboxylesterase (E.C. 3.1.1.1) previously identified as a target for reactive metabolites of halothane. The extent to which covalent modification of this protein by reactive metabolites contributes to the production of hepatotoxic effects remains to be determined.

Introduction Many potent cytotoxins act by interfering directly with important biochemical processes either by means of enzyme inhibition or through interaction with pharmacological receptors. However, many compounds which elicit toxic responses have little chemical or pharmacological reactivity until they undergo biotransformation to chemically reactive intermediates (1-3). Unless detoxified by further biotransformation, these intermediates may react nonenzymatically with cellular constituents leading to their “denaturation” through covalent binding and/or autoxidative damage. When the chemical defense and repair capacities of a cell are exceeded, such biochemical insults may be followed by a cascade of secondary events which amplify and/or broaden the initial response, thus compromising the cell further and leading to observable toxic responses. The first simple organic compound whose cytotoxicity was clearly associated with covalent binding of reactive metabolites is bromobenzene. Brodie et al. showed that during the biotransformation of bromobenzene in vitro, reactive metabolites form and become covalently attached to microsomal proteins (4). They also demonstrated that in vitro, glutathione can intercept these reactive metabolites and prevent their covalent binding to proteins. In vivo, both the time course of formation and the histological distribution of covalently bound metabolites correlate with the degree of liver injury (5, 6). Previous studies in our laboratory showed that both epoxide and quinone metabolites of bromobenzene bind covalently to rat liver proteins under toxicologically relevant conditions in vivo (7-9), as well as in precision-cut liver slices (10). How* Address correspondence to this author: tel, 785-864-3750; fax, 785864-5326; e-mail, [email protected].

ever, it remains unclear as to which types of reactive metabolites or binding events are most significant toxicologically (11, 12). More recently attention has turned from structural identification of adduct moieties to the identification of the cellular proteins which become adducted and the effect of adduct formation on their ability to carry out their original biochemical role in the cell. A number of cytosolic, microsomal, and mitochondrial proteins have been shown to be targets for covalent binding by reactive metabolites of halothane, acetaminophen, tienilic acid, S-(haloalkyl)cysteine derivatives, diclofenac, and others. In this paper we report that a liver microsomal esterase previously shown (13) to be a target for reactive metabolites of halothane is also a target for the covalent binding of bromobenzene metabolites.

Experimental Procedures Chemicals and Biochemicals. [14C]Bromobenzene was prepared from [14C]aniline by a sequence of bromination, diazotization, and reduction as described previously (14). Electrophoresis supplies were from BioRad. All other chemicals and biochemicals were obtained from reputable commercial sources and used as received. Animals and Microsome Preparation. Male SpragueDawley rats (180 g; Charles River Laboratories, Wilmington, MA) were housed in a temperature- and humidity-controlled room with a 12-h light/dark cycle and ad libitum access to food and water. After acclimating for at least 3 days, animals were given daily ip injections of sodium phenobarbital (80 mg/kg) in 0.9% saline (1.0 mL/kg). After the third dose, food was withheld overnight, and the next morning the rats were killed by decapitation under CO2 narcosis. Their livers were removed, chilled, and homogenized in ice-cold 50 mM potassium phosphate buffer, pH 7.4, containing 0.15 M KCl, 1 mM EDTA, 0.5 mM DTT (4 mL/g of tissue). The homogenate was successively

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A Target Protein for Bromobenzene Metabolites centrifuged at 3000g (10 min), 12000g (20 min), and 100000g (60 min), and the microsomal pellet was homogenized in 0.1 M sodium pyrophosphate buffer, pH 8.2 (2 mL/g of tissue), followed by recentrifugation at 100000g. The final microsomal pellet was resuspended in 100 mM potassium phosphate, pH 7.4, 20% glycerol, 0.5 mM EDTA (0.2 mL/g of tissue) and stored at -70 °C. The protein content of the microsomal preparation was determined by Bradford assay (15), and the cytochrome P450 and b5 contents were determined by difference spectra according to Omura and Sato (16). Conduct of Incubations. Incubations were carried out in 250-mL glass-stoppered Erlenmeyer flasks for 90 min at 37 °C in a shaking water bath. They contained 100 mg of microsomal protein, 100 µmol of [14C]BB (5.17 Ci/mol, delivered in 1.0 mL of acetonitrile), and a freshly prepared NADPH-generating system (consisting of 50 µmol of NADP, 500 µmol of glucose-6phosphate, and 50 units of glucose-6-phosphate dehydrogenase predissolved in 0.5 mL of incubation buffer), all in a final volume of 50 mL of 100 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA. Incubations were terminated by chilling the incubation flask in an ice bath. The microsomes were separated from the incubation mixture by centrifugation at 100000g and washed to remove unbound label by two cycles of homogenization in 50 mM sodium phosphate, pH 7.4, followed by centrifugation at 100000g. Protein Purification. The washed microsomal pellet was resuspended in 10 mL of 50 mM sodium phosphate, pH 7.4, and the suspension stirred on ice while sufficient sodium cholate stock solution (20%, w/v) to achieve a final cholate concentration of 3.3% was added dropwise. After stirring for 20 min the sample was then diluted with buffer to a final cholate concentration of 2% (w/v), after which insoluble material was removed by centrifugation at 100000g for 1 h. The supernatant obtained after centrifugation (ca. 16 mL) was divided into two aliquots of 8 mL each; one was frozen for later use and the other was dialyzed against 100 mM Tris, pH 7.4, 0.1% Triton X-100 (5 × 1000 mL). The last two buffer changes removed little radioactivity compared to the first three. Ion-Exchange Chromatography. The dialyzed sample was centrifuged at 100000g, and the resulting clear supernatant was loaded at a flow rate of 2 mL/min onto a 5-mL High Q column (BioRad) which had been equilibrated with 100 mM Tris, pH 7.4, 0.1% Triton X-100. The column was washed with equilibration buffer (120 mL) until the radiolabel in the eluate was less than or equal to twice the background. Retained proteins were then eluted with a gradient of NaCl (0-1 M over 120 mL). Column fractions (2 mL) were pooled based on their content of radiolabel and protein (see below), and the pooled fractions were concentrated by ultrafiltration using a PM-10 membrane (Amicon) and stored at -20 °C until used. Preparative Electrophoresis. The proteins in the pooled High Q column fractions were further purified by preparative electrophoresis using a BioRad model 491 prep cell. The sample (1 mL) was diluted with an equal volume of 130 mM Tris, pH 6.8, containing 2% (w/v) SDS, 200 mM DTT, 20% (v/v) glycerol, 0.04% (w/v) bromophenol blue and heated for 5 min in a boiling water bath. After cooling, the sample was loaded at 1 mL/min onto a preparative PAGE column (28-mm diameter × ca. 100mm length) containing a 7% resolving gel (30 mL) and a 4% stacking gel (5 mL). The electrophoresis was run at a constant power of 12 W, and successive 2.5-mL fractions were collected and later pooled according to their content of protein and radioactivity (see below). Pooled fractions were concentrated by ultrafiltration and stored at -20 °C. Analysis of Protein Fractions. During protein fractionation radiolabel was monitored by scintillation counting of sample aliquots. Protein eluting from the High Q column was determined by measurement of A280 with a Cary 118 spectro1 Abbreviations: DTT, dithiothreitol; GST, glutathione transferase; PAGE, polyacrylamide gel electrophoresis; PVDF, poly(vinylidene difluoride); SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid.

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Figure 1. Outline of the separation scheme used for isolation of microsomal proteins adducted by metabolites of [14C]bromobenzene. photometer. Aliquots (20 µL) of fractions from preparative elecrophoresis were individually analyzed by SDS-PAGE (17) on a 4-20% gradient minigel (BioRad) with silver staining of the gels. To locate radiolabeled proteins, aliquots from concentrated pooled fractions were separated by SDS-PAGE and transferred electrophoretically to PVDF membranes. The membranes were then stained for protein with 0.4% Coomassie brilliant blue R-250 in methanol/acetic acid/water (80:5:15, v/v), destained in the same solvent and subjected to phosphorimaging analysis for detection of 14C using a Molecular Dynamics storage phosphor screen, scanning unit, and software. N-Terminal Sequence Analysis. For N-terminal sequence analysis, an aliquot (500 µL) of the pooled fractions from preparative electrophoresis was precipitated by addition of 500 µL of 20% TCA and centrifugation. The pellet was acetonewashed and solubilized by addition of 130 mM Tris, pH 6.8, containing 2% (w/v) SDS, 200 mM DTT, 20% (v/v) glycerol, 0.04% (w/v) bromophenol blue followed by heating for 5 min in a boiling water bath. The proteins in the sample were separated by SDS-PAGE on a 12% minigel with a 4% stacking gel and electrophoretically transferred to PVDF membranes. The immobilized protein was visualized by Coomassie staining, and the radiolabel was detected by phosphorimage analysis as described above. Finally, portions of the membrane containing protein bands of interest were excised and submitted to automated N-terminal sequence analysis using a Perkin-Elmer/ Applied Biosystems 473A sequencer. The N-terminal sequence was compared to sequences of known proteins by searching the Genbank protein data bank (18).

Results After incubation of rat liver microsomes with [14C]BB and an NADPH-generating system, the microsomes were recovered and radiolabeled proteins solubilized and isolated as outlined in Figure 1. A majority of the

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14C-adducted

Figure 2. Separation of proteins by High Q anion-exchange chromatography: b, dpm/100-µL aliquot of fractions; O, protein content (A280) of individual 2-mL fractions. The solid line represents the linear NaCl gradient (0-1.0 M) from fractions 60-120.

unreacted bromobenzene and its soluble metabolites were removed by three cycles of centrifugation and resuspension of the microsomes. After solubilization with cholate, centrifugation, dialysis, and recentrifugation removed additional radiolabel equivalent to 2.0%, 9.6%, and 2.8% of the original radioactivity, respectively. Examination of the solubilized microsomal proteins by SDS-PAGE showed several nearly overlapping bands of protein around 50-60 kDa and another prominent band near 70 kDa (see below). The resulting protein mixture was fractionated by High Q anion-exchange chromatography. Figure 2 depicts the elution profiles observed for 14C and protein, as well as the NaCl gradient used. Two distinct peaks of radioactivity were eluted from the column in the loading flowthrough and the subsequent wash with equilibration buffer (fractions 1-59). Although it was not possible to measure protein (A280) in fractions 1-18 because of differences in the concentration of Triton X-100 of the sample loaded vs the elution buffer, a significant protein peak was observed to elute with the second radiolabel peak in the late flow-through fraction. More strongly retained proteins were eluted with a NaCl gradient beginning with fraction 60. Both radiolabel and protein were detected eluting from the column in two overlapping wide peaks. On the basis of the radiolabel and protein peak profiles shown in Figure 2, High Q column fractions 7-13, 14-29, 70-74, and 75-93 were combined to form pools 1-4, respectively, which contained 8.5%, 21.7%, 5.4%, and 33% of the radioactivity originally applied to the column (69% total recovery). The proteins in these pools were analyzed by SDSPAGE followed by transblotting, staining for protein, and phosphorimaging of the blots to detect radiolabel. As shown in Figure 3, the majority of the proteins in the pooled column fractions ranged from 45 to 70 kDa. Pools 2-4 contained additional protein bands in the 20-kDa range. Many of the protein bands also contained some radiolabel, but the apparent labeling density of the individual bands was rather variable. Most notably, pool 3 (lane 3 in Figure 3) has two major protein bands, only one of which is radiolabeled, and there is an additional small 14C-labeled protein band appearing immediately below the more pronounced protein band around 20 kDa in the same lane. In pool 1 (lane 1 in Figure 3), only two of the three major proteins are radiolabeled, while lane 2 contains a broad band of radioactivity but relatively little protein in the very low-molecular-weight area. To identify individual radiolabeled proteins, further purification was clearly needed, but since it was not clear

Rombach and Hanzlik

Figure 3. SDS-PAGE analysis of pooled fractions from High Q anion-exchange chromatography: panel A, Coomassie stained proteins after separation on a 4-20% gradient gel and transfer to a PVDF membrane; panel B, results of phosphorimaging of the same membrane shown in panel A. Lanes are labeled as follows: S, standards (molecular weight markers); M, solubilized microsomal proteins before High Q chromatography; lanes 1-4 correspond to High Q pools 1-4 (see Figure 1 and text for details).

Figure 4. Elution of radiolabel during preparative PAGE of High Q pool 1. Each point represents the dpm 14C present in a 0.5-mL aliquot of a 2-mL column fraction. Vertical bars indicate fractions combined into pools 1A-1C.

at this point which of the radiolabeled proteins was of greatest relevance to bromobenzene-mediated hepatotoxicity, we pursued purification of several of the most abundant radiolabeled proteins from several of the High Q column fraction pools. Because many of the proteins in the pooled samples appeared to separate well on SDSPAGE (Figure 3), we fractionated an aliquot (18%) of pool 1 by means of preparative PAGE; samples from selected fractions were analyzed by scintillation counting and by SDS-PAGE and silver staining. As shown in Figure 4, a majority of the radioactivity was found in early fractions which contained relatively little protein. However, a single band of protein eluted in association with a small tail observed on the initial major radioactive peak (fractions 25-39), while another single band of protein of higher MW eluted concurrent with a second 14C peak in fractions 40-60. Finally, a single band of protein was detected in fractions 71-87, but little radiolabel was associated with this fraction. On the basis of the analytical SDS-PAGE results, fractions 25-39, 44-55, and 7187 from the preparative electrophoresis were combined to form pools 1A, 1B, and 1C, respectively, which contained 4%, 10% and a trace of the radioactivity originally applied (92% recovery overall). Figure 5 shows a silver-stained SDS-PAGE of these three pooled fractions as well as the original unseparated mixture. Each pool appears to contain a single sharp band of protein. A comparison of the protein patterns in pool 1 (lane 1 of Figure 3 or 5) vs pool 1B (lane 1B of

A Target Protein for Bromobenzene Metabolites

Figure 5. SDS-PAGE analysis of pooled fractions from preparative PAGE separation of proteins in pool 1 from High Q chromatography. Samples were separated by SDS-PAGE (10% gel) and silver-stained. Lane 1 shows pool 1 from the High Q column before prep-PAGE; lanes 1A-1C show the corresponding pools from prep-PAGE fractions (see Figures 1 and 4 and text for details).

Figure 6. SDS-PAGE analysis of pool 1B from prep-PAGE separation of High Q pool 1. An aliquot of pool 1B (1800 dpm total) was treated with TCA and the precipitate washed, solubilized, separated by SDS-PAGE (12% gel), and transferred to a PVDF membrane. The membrane was Coomassie stained for protein (left) and submitted to phosphorimaging analysis (right). Molecular weight markers are shown at the far left.

Figure 7. Amino acid sequence found for the major protein band shown in Figure 6. The published sequences for hydrolases A and B are shown for comparison. See text for details.

Figure 5) suggests that pool 1B contains a majority of the radiolabeled protein originally contained in High Q column pool 1. As shown in Figure 6, this protein sample, even when overloaded, contains one major protein band at approximately 57-59 kDa (Figure 6, left side), and this band is strongly radiolabeled (Figure 6, right side). To obtain protein suitable for N-terminal microsequencing, material from pool 1B was run on SDS-PAGE, transblotted, stained, and phosphorimaged (as in Figure 6), after which the large densely stained protein band was excised from the blot for direct N-terminal sequence analysis. The results are summarized in Figure 7. The first 18 turns of Edman analysis yielded unambiguous assignments in 13 positions, but three turns (numbers 1, 9, and 11) yielded two amino acid peaks each, and two turns yielded no assignable amino acid peaks. A search of the Genbank (18) data bank for similar sequences found one match, with 100% identity, to the known sequence of a 57-kDa rat liver microsomal carboxylesterase known as hydrolase A (19, 20). The appearance of two amino acids in turns 1, 9, and 11 of the sequencing

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Figure 8. SDS-PAGE analysis of pools 1 and 1B. Aliquots of pools 1 (3000 dpm) and 1B (345 dpm) were treated and the precipitates washed, separated by SDS-PAGE (7.5% gel), and transferred to a PVDF membrane. The latter was Coomassie stained for protein (left panel) and submitted to phosphorimaging (right panel). Molecular weights are shown at the left; the two bands labeled A and B correspond to 57 and 59 kDa, respectively.

reported in Figure 7 can easily be explained by the presence of the closely related 59-kDa isozyme, hydrolase B. To ascertain whether hydrolase A or hydrolase B was radiolabeled, samples of pools 1 and 1B were reanalyzed by SDS-PAGE under conditions optimized for separating these isozymes. The electrophoretically separated proteins were then transblotted to a PVDF membrane, Coomassie stained, and phosphorimaged. To confirm the identification of these two isozymes, the individual 57and 59-kDa bands were then excised from the blot and analyzed by N-terminal sequencing. The Coomassie staining results (Figure 8, left panel) show a major protein band at 57 kDa and a smaller distinct band at 59 kDa. The phosphorimaging results clearly show that these two bands, and only these two bands, contain radiolabel. Interestingly, the major protein band contains the lesser amount of radiolabel, while the minor protein band contains the majority of the radiolabel. Sequencing results (not shown) confirmed that the major band at 57 kDa is hydrolase B while the minor protein band is hydrolase A. No evidence of other contaminating proteins was detected in the sequencing of these two bands. Unfortunately, attempts to examine the proteins in pool 1 by means of 2D-gel electrophoresis were thwarted by the poor solubility behavior of the 57- and 59-kDa proteins in the first (isoelectric focusing) direction. Finally, to evaluate whether the covalent labeling of these enzymes by bromobenzene metabolites adversely affected their catalytic activity, we incubated liver microsomes from phenobarbital-induced rats with bromobenzene and an NADPH-generating system in vitro, reisolated the microsomes by centrifugation, and evaluated their ability to catalyze the hydrolysis of 1-naphthylacetate and p-nitrophenylacetate (19). For controls we used microsomes treated similarly but with bromobenzene omitted as well as microsomes not preincubated. With each substrate, the results obtained with the three types of microsomes were identical (data not shown).

Discussion The acute hepatotoxic effects of bromobenzene are strongly associated with its biotransformation and the attendant covalent binding of reactive metabolites to hepatocellular proteins. Similar correlations have also been observed for other simple organic molecules including chloroform (21), halothane (22), chloramphenicol (2325), thioacetamide (26), and thiobenzamide (27) (which

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undergo oxidation to acylating species), as well as for naphthalene (28), acetaminophen (29), and various phenolic derivatives (30, 31) (which undergo oxidation to electrophilic quinone, quinonimine, or quinone methide species). The diversity of reactive metabolites derived from these agents suggests that if their covalent binding contributes causally to cell injury, the identification of the proteins which become modified may be of greater relevance to understanding toxicological mechanisms than the identification of the various small molecules which bind to them. Experience in several laboratories has indicated that protein adduct formation is a selective process, i.e., that not all cellular proteins are equally labeled by reactive metabolites. In this context the two most extensively studied hepatotoxicants are halothane and acetaminophen. Halothane causes a very mild form of liver injury in susceptible individuals and, in rare cases, a much more severe form of hepatitis that may progress to fulminant liver failure and death (13, 22, 32, 33). Considerable evidence suggests the severe reaction has an immunological component, but the formation of protein adducts of halothane metabolites and the subsequent formation of antibodies to these neoantigens appear to be integral to the process. Among the proteins which are trifluoroacetylated during halothane metabolism are a microsomal carboxylesterase (13), a phenobarbital-inducible form of cytochrome P450 (34), calreticulin (35), protein disulfide isomerase (36), and UDP-glucose: glycoprotein glycosyltransferase (37). Acetaminophen also causes centrolobular necrosis in several species of laboratory animals and humans. Although there is no evidence supporting an immunological mechanism of cell injury with acetaminophen, many proteins adducted by acetaminophen metabolites are readily recognized by antibodies raised against synthetic haptens related to acetaminophen (38, 39). Acetaminophen target proteins which have been identified include the cytosolic proteins glutathione transferase (40), 56-58-kDa selenium-binding protein(s) of unknown function (41, 42), and N10-formyltetrahydrofolate dehydrogenase (43); the microsomal protein calreticulin and two isozymes of protein disulfide isomerase (44); and the mitochondrial proteins glutamine synthase (45), glutamate dehydrogenase (46), aldehyde dehydrogenase (47), and carbamoylphosphate synthetase I (48). The reactive metabolite of acetaminophen shows considerable preference for reaction with protein sulfhydryl groups (49, 50) but also binds to other sites (44). It may be significant that a majority of the acetaminophen target proteins identified to date are sulfhydryl proteins. At the level of reactive metabolites, bromobenzene generates considerably greater diversity than halothane or acetaminophen. Among its reactive metabolites are two arene oxides and several quinones and bromoquinones, all of which have been observed to bind to protein sulfhydryl groups (8, 9) and other nucleophiles (51). Thus far only two hepatic proteins have been identified as targets for reactive metabolites of bromobenzene: subunit 1 of the cytosolic GST gene family proteins (52) and the carboxylesterase isozymes reported herein. The cytosolic GSTs exist as homo- and heterodimers of several different subunits having one or more free sulfhydryl groups (53, 54). Modification of these groups by chemical derivatization or protein engineering has no general detrimental effect on catalytic activity unless the

Rombach and Hanzlik

-SH group is close enough to the glutathione binding site that its covalent modification blocks GSH binding (55, 56). The microsomal carboxylesterase originally identified as a target for halothane metabolites (E.C. 3.1.1.1) was subsequently shown to consist of two isoforms, only one of which (hydrolase B, the minor isozyme comprising 0.5% of microsomal protein) contains a free sulfhydryl group. Interestingly, this minor isozyme becomes much more extensively labeled than the ca. 3-fold more abundant hydrolase A. Unfortunately we do not yet know what type(s) of metabolite(s) is involved in labeling these hydrolase isozymes; answers to these question are currently being sought. It is noteworthy, however, that of the dozen or so proteins identified as targets of reactive metabolites, several are now known to be targets for two or more hepatotoxicants. The multiple involvement of these proteins might be only coincidental. For example, it could be that these proteins are sufficiently abundant and reactive toward electrophiles that their covalent modification by reactive metabolites generated in situ is kinetically inescapable. However, in view of the known enzymatic functions of these proteins, it is hard to conceive of their covalent modification as being particularly harmful to liver cells. On the other hand, it is not entirely inconceivable that these proteins may have important but as yet undiscovered roles in living cells and that their alkylation by different hepatotoxicants may be signaling the existence of a common pathway from reactive metabolite generation to the generation of toxic responses.

Acknowledgment. We thank the Kansas State University Biotech Core Facility for their assistance in protein sequencing. Financial support for this work was provided in part by NIH Grant GM-21784.

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