Chem. Res. Toxicol. 1995,8, 729-735
729
Bromobenzene 3,4-0xide Alkylates Histidine and Lysine Side Chains of Rat Liver Proteins in Vivo Ramesh B. Bambal and Robert P. Hanzlik" Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045-2506 Received February 9, 1995@
The hepatotoxic effects of bromobenzene (BB) are correlated with and generally ascribed to the covalent modification of cellular proteins by chemically reactive metabolites, particularly BB-3,koxide. Previous studies revealed that quinone a s well a s epoxide metabolites of BB form adducts to protein sulfur nucleophiles, that the quinone-derived adducts are more abundant by a factor of ca. 7, and that collectively these sulfur adducts account for only about 10% of the total protein covalent binding [Slaughter, D. E., and Hanzlik, R. P. (1991) Chem. Res. Toxicol. 4,349-3591. To examine the possibility that metabolically-formed BB-3,4-oxide alkylates nitrogen nucleophiles on proteins under toxicologically relevant conditions in vivo, we synthesized standards of iV4pbromophenyl)histidine ( 7 )and N'-(p-bromophenyl)lysine (8) a s anticipated adduct structures and used them to guide a chromatographic search for their presence in hydrolysates of liver protein from BB-treated rats. While radio-LC chromatography and GC/MS provide unequivocal evidence for their presence, the amounts of 7 and 8 observed are very low (el% of total covalent binding). The apparently small net contribution of epoxide metabolites to covalent binding of BB in vivo suggests the majority of binding may arise via quinone metabolites, but this should not be construed to imply that quinone adducts are necessarily more important toxicologically than epoxide adducts; in this context the identity of the protein targets is probably a t least a s important as the type of electrophilic metabolite involved.
Introduction Aryl halides such as bromobenzene (BBY are known to cause organ-selective tissue injury in mammals, particularly to liver (1-3) kidney (41, and lung (5). The hepatotoxicity of BB has been extensively studied as a model for cellular injury induced by chemicals and has been strongly associated with and generally attributed to the formation of chemically-reactive metabolites which then covalently bind to cellular protein nucleophiles (3, 6-8). Adducts of BB metabolites to sulfur nucleophiles of rat liver proteins have been shown to arise from several different reactive metabolites, including epoxides 1 and 2 and quinones 3-5, under toxicologically relevant conditions in vivo (9, IO). The first BB-protein adduct to be identified was S-(p-bromophenyl)-L-cysteine(6) (9).However, adduct 6, along with minor amounts of its ortho and meta isomers, accounted for only 1-2% of the total covalent binding of [l4C1BBto rat liver proteins. More recently, eight adducts of BB-derived quinone metabolites 3-5 to protein sulfur nucleophiles were identified by GCI MS after their conversion to thioanisole derivatives by alkaline permethylation (IO). These adducts represented nearly 7% of the total covalent binding of [l4C1BBto rat liver protein. Altogether, the covalent binding of BB epoxides 1 and 2 and quinones 3-5 to protein sulfur nucleophiles accounts for only about 8-9% of the total covalent binding of BB metabolites under toxicologically relevant conditions in vivo. Therefore, other protein nucleophiles
* Address correspondence to this author. Tel: 913-864-3750;fax: 913-864-5326. @Abstractpublished in Advance ACS Abstracts, June 1, 1995. Abbreviations: BB, bromobenzene; RIC, reconstructed ion chromatogram; RLP, rat liver protein; TIC, total ion chromatogram.
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must also be involved in adduct formation. Indeed, in vitro studies have implicated histidine residues as targets for reactive metabolites of P4C1BB ( I I ) , although the structures of the adducts formed and the reactive metabolites responsible were not identified. Histidine has also been found to be a target for alkylation by benzo[alpyrene diol epoxide metabolites (121, as well as by 4-hydroxynonenal(13)and quinone methide metabolites (14).Lysine side chains are highly basic and extensively protonated in vivo; nevertheless, a number of reactive chemicals are known to alkylate protein-lysine side chains under physiologically relevant conditions (15-191. A plausible mechanism for the alkylation of protein nitrogen nucleophiles by epoxide 1, which is suspected to be toxicologically the most significant reactive metabolite of BB (ZO), is shown in Scheme 1. Epoxide ringopening followed by elimination of water from cyclohexadienol adduct 9 gives bromophenyl adduct 10. According to Scheme 1, reaction of epoxide 1 with protein cysteine, histidine, or lysine side chains, followed by protein hydrolysis, would yield hydrophobic amino
Q893-228X/95l27Q8-Q729$09.Q~~Q 0 1995 American Chemical Society
Bambal and Hanzlik
730 Chem. Res. Toxicol., Vol. 8, No. 5, 1995 Scheme 1 6000 I
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acids 6-8. Among these, only cysteine adduct 6 has been positively identified (9). The objective of the present study was to evaluate the possible covalent binding of 1 to histidine andlor lysine side chains of rat liver protein in vivo, and to estimate the contributions of these events to the overall covalent binding of BB metabolites under toxicologically relevant conditions. In this paper we present evidence for the isolation, unambiguous identification, and quantitation of 7 and 8 from hydrolysates of liver protein from BB-treated rats.
Experimental Procedures Instrumentation. GC/MS analyses were performed on a Hewlett-Packard 5890 gas chromatograph equipped with a mass selective detector ( H P 5971A) and a n autoinjector ( H P 7673) and controlled by the HP-Unix based Chem Station software. Samples (5 ,uL) were injected in the splitless mode and analyzed on a DB-5 column (0.26 mm x 30 m; J & W Scientific, Folsom CAI. The injection port and G C N S interface were maintained at 290 "C, and the oven temperature was programmed as follows: 100 "C for 2 min, then 20 "C/min to 200 "C, hold for 1 min, then 5 "Cimin to 250 "C, and hold for 15 min. Electron ionization (70 eV) mass spectra were recorded over the mass range 40-650 amu a t a scan rate of 0.97 scads. LC analyses were performed using a Shimadzu system with dual LC-6A pumps, a n SCL-6A controller, an SPD-6A variable wavelength detector operated a t 250 nm, and a C-18 reverse phase column (Alltech Econosil, 10 ,pm, 4.6 x 250 mm) eluted with 0.05 M ammonium acetate/acetonitrile (80:20 v/v) at 1m I J min. Under these conditions the retentions times of 6, 7, and 8, synthesized as described elsewhere (21,221,were ca. 11, 20, and 22 min, respectively. LC fractions were collected and counted for 14C on a Packard 1900 TR liquid scintillation analyzer. Isolation of BB-Adducted Rat Liver Protein. As described previously (9,10,23),14 male Sprague-Dawley rats (223 i 12 g; Sasco, Inc., Omaha, NE) were injected ip with sodium phenobarbital in 0.9% saline (50 mg/kg, 2.2 m m g ) for 3 consecutive days. After the third dose, food but not water was withheld overnight, and the next morning six rats were dosed ip with [U-l4C1BB(2.5 mmoL'kg, 0.33 CUmol, 5 mmolimL in corn oil). The remaining eight rats were similarly dosed with nonradioactive BB. Four hours after dosing, the rats were killed by COz narcosis and their livers removed and chilled i n ice-cold buffer (0.1 M potassium phosphate, pH 7.4, containing 1 mM EDTA and 1.12% KC1). Livers from individual rats (average weight 9.6 & 0.7 g) were homogenized individually in the same buffer (4 mL/g liver) using a Polytron homogenizer. A 10 mL aliquot of each individual homogenate was combined to create a common pool of homogenate for use in pilot studies; otherwise, homogenates from individual rats were kept separate. Homogenates were treated with 1.5 volumes of 50% trichloroacetic acid, and the precipitated protein was washed, dried, and isolated as a free-flowing off-white powder (9, 10, 24). By a similar procedure, 11.9 g of liver protein was obtained from the pooled homogenate of eight rats treated with nonradioactive BB.
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Figure 1. Typical XAD-2 chromatogram of hydrolysate of liver protein from a rat treated with [l4C1bromobenzene. Fractions were pooled into five groups (A-E) for further analysis.
Protein Hydrolysis. In a typical experiment, [l4C1RLP(500 mg) and 50 mL of 6 M HCl were heated for 24 h under a reflux condenser and a nitrogen atmosphere in a n oil bath maintained a t 120 "C. The resulting brown solution was cooled and filtered through a glass frit to remove a small amount of tar (5 mg) which was found to contain only 1%of the original I4C. The dark brown filtrate was extracted with ether ( 5 x 10 mL) and the pooled ether extract evaporated, yielding a small brown residue (2 mg; 7% of the original 14C). The aqueous hydrolysate was concentrated on a rotary evaporator (25-40 Torr, 90-95 "C bath), yielding a viscous brown residue containing, inter alia, hydrochloride salts of protein-derived amino acids. Control experiments showed that 6-8 could be recovered quantitatively after being exposed to protein hydrolysis conditions. Fractionation of Protein Hydrolysates. The residue from hydrolysis of 500 mg of liver protein from rats treated with [l4C1BB was dissolved in 4.5 mL of water and loaded onto a column of XAD-2 resin (50 g; 30 x 200 mm) which had previously been washed with several column volumes of methanol and then water. Based on previous experience with 6 (9)and preliminary experiments with 7 and 8, the XAD-2 column was sequentially eluted with water (170 mL), 20% methanollwater (170 mL), methanol (100 mL), and 10% acetic acidmethanol (50 mL). The eluate was collected in 100 5 mL fractions, and a 1 mL aliqout of each was counted for radioactivity. After plotting the radiochromatogram, individual fractions were combined into five pools designated A-E as illustrated in Figure 1. Thus pools A and B were eluted with water, pool C with 20% methanoywater, pool D with methanol, and pool E with 10%acetic acidmethanol. Under these conditions, representative hydrophobic amino acids, including phenylalanine, tryptophan, and compounds 6-8, elute quantitatively i n pool D; in contrast, amino acids with small a n d o r polar side chains, including proline, valine, glutamic acid, histidine, and lysine, are neither retained nor retarded by the column and elute in pool A. Nonradioactive BB-adducted r a t liver protein (3.0 g) was similarly hydrolyzed in 330 mL of 6 M HCl and the hydrolysate processed by filtration, extraction with ether, concentration, and fractionation over XAD-2 resin (150 g i n a 35 x 350 mm column). The column was sequentially eluted with 600 mL of water (pools A and B), 600 mL of 20% methanovwater (pool C), and 600 mL of methanol (pool D). LC Analysis of Pooled XAD-2 Fractions. Each pool of fractions from XAD-2 chromatography of radioactive hydrolysate was concentrated under vacuum, the residues were reconstituted in 0.3-1.0 mL of water, and an aliquot (50 pL) was subjected to reverse-phase HPLC analysis. Eluents were collected in 1 mL fractions, and each fraction was counted for I4C. A representative radiochromatogram of pool D, which is the XAD-2 fraction in which hydrophobic amino acid standards of 7 and 8 elute, is shown in Figure 2. Preparative LC Fractionation of Material in Pool D. The solvent from XAD-2 pool D was concentrated under vacuum,
Chem. Res. Toxicol., Vol. 8, No. 5, 1995 731
BB-3,4-oxideAlkylates Protein N-Nucleophiles 1601
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Figure 2. HPLC chromatograms of material in hydrolysate pool D (panel a ) and standards of hydrophobic amino acids including phenylalanine and compounds 6-8 (panel b; absorbance values are on a relative scale). and the residue obtained (37 mg from 3.0 g of protein) was reconstituted in 2 mL of methanol. Multiple injections of 50 pL aliquots of pool D material were made, and collection of the eluents at retention times corresponding to standards of 7 and 8 yielded, after evaporation, 920 and 580 pg, respectively, of pale brownish residues. Derivatization and GC/MS Analysis. Individual dry residues of fractions obtained from preparative HPLC of XAD-2 pool D were dissolved in 1 mL of n-BuOH saturated with HC1 gas (ca. 3 M) and heated in a sand bath at 100 "C in a 13 x 100 mm culture tube sealed with a Teflon-lined screwcap for 15 min (25). (Caution: Because pressure might develop, reactions were conducted behind a safety shield in a fume hood and tubes were cooled prior to opening.) The excess BuOH/HCl was removed under vacuum, and to the dry residue thus obtained was added 0.5 mL of dichloromethane and 0.5 mL of trifluoroacetic anhydride. The tubes were heated by partial immersion in a sand bath a t 150 "C for 5 min (see Caution statement above). Excess solvent was removed in vacuo and the residue reconstituted in 20 p L of methanol for G C M S analysis. Standards of 7 and 8 were derivatized in a similar fashion for G C M S analysis.
Results and Discussion After treatment of phenobarbital-induced rats with [14C]BB,yields of isolated liver protein (2.29 f 0.24 ghat) and covalent binding label densities (2.9 f 1.4 nmol equiv of BB/mg of protein) were comparable to those reported previously (9, 10). Also in agreement with previous experience (9), the vast majority of protein-bound 14C remained water soluble and nonextractable by ether after protein hydrolysis. For further analysis, hydrolysates of liver protein (500 mg) from three individual rats were processed separately but identically. Fractionation of each hydrolysate over XAD-2 resin using a scheme
optimized for separating hydrophobic from nonhydrophobic amino acids produced intriguing chromatograms, of which Figure 1 is representative. The first few fractions (pool A) contained material that was neither retained nor retarded by the column. These fractions contained 80-90% of the mass and 21-23% of the radioactivity originally applied to the columns. Undoubtedly, much of this material is comprised of the hydrochloride salts of nonhydrophobic amino acids released during protein hydrolysis. Pool B contained hydrolysate material which was retarded but not retained by the column during elution with water and accounted for 4-7% of the mass and 13-28% of the 14C originally applied to the column. Upon rechromatography over XAD-2, material from pools A and B eluted discretely as originally isolated, showing relatively little cross contamination or interconversion. Since further elution with water was unproductive, elution with 20% methanol was begun. This brought off ca. 1%of the mass and 6-13% of the radioactivity applied to the column. Control experiments showed that 20% methanol does not elute hydrophobic amino acids such as phenylalanine or 6-8, whereas pure methanol elutes them rapidly and quantitatively. Changing the elution solvent to pure methanol eluted a peak containing 1734% of the radioactivity but only 1%of the mass originally applied (pool D, Figure 1). Finally, elution with methanovacetic acid brought off a minute amout of material containing 7-27% of the originally applied radioactivity. The overall recoveries of mass and radioactivity from the XAD-2 columns were 95-105% and 81119%, respectively. Further characterization of the materials in XAD-2 fraction pools A-E was persued via LC using a C-18 reverse-phase column and a n elution scheme optimized for the separation of standards of phenylalanine and compounds 6-8, as shown in Figure 2b. Under these conditions the 14C materials contained in pools A-C nearly all eluted at retentions times shorter than that of phenylalanine (data not shown); hence these fractions were not pursued further. Pool E material was similarly ignored, because based on results with standards and the elution profile of 14Cin the pool D region of Figure 1, it could not possibly contain 7 or 8. The LC radiochromatogram of pool D material is shown in Figure 2a. Peaks of radioactivity eluting a t retention times corresponding to 6 and 7 are clearly visible in bands labeled X and Y, respectively. Since 6 was thoroughly characterized as an adduct of BB to rat liver protein in an earlier investigation (9), it was not pursued further. For confirmation that the peak of radioactivity in band Y of Figure 2a was indeed N'-@bromophenyl)histidine (71, and to search for the possible presence of N'-(pbromopheny1)lysine (8)in band Z of Figure 2a, we turned to GC/MS. For this, pooled liver protein from rats treated with nonradioactive BB was processed via acid hydrolysis and XAD-2 chromatography to obtain pool D, which was further processed by preparative C-18 HPLC to obtain bands Y and Z. Eluents collected under bands Y and Z were lyophilized and the residues treated to convert amino acids to their N-trifluoroacetyl n-butyl esters and analyzed by GC/MS with data collection in the fullspectrum mode. A portion of the total ion chromatogram of derivatized band Y material is shown in Figure 3a. After derivatization, authentic 7 has a retention time of ca. 25.5-26 min and a mass spectrum as shown in Figure 4a.
Bambal and Hanzlik
732 Chem. Res. Toxicol., Vol. 8, No. 5, 1995
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“spectroscopic signature” for 7 and generated a reconstructed ion chromatogram (RIC)by summing the intensities of these six ions vs time. As shown in Figure 3b, this RIC shows a single strong peak at the correct retention time for derivatized 7. An averaged backgroundsubtracted mass spectrum collected just under the peak shown in Figure 3b is displayed in Figure 4b. The excellent agreement of this spectrum with that of the standard in Figure 4a provides unequivocal evidence that 7 is indeed present, albeit at low levels, in the hydrolysate of liver protein from BB-treated rats. A search throughout the entire GCIMS chromatogram of derivatized band Y failed to reveal the presence of any other brominecontaining materials, although small amounts could easily have been missed under large quantities of nonbrominated materials. A similar strategy was adopted for searching band Z material for 8 by GC/MS. A portion of the total ion chromatogram of derivatized band Z material is shown in Figure 5a. After derivatization, authentic 8 has a retention time of ca. 20.5-21 min and a mass spectrum as shown in Figure 6a, where bromine-containing molecular and fragment ions are conspicuous at m / . 267, 269,280,282,474,476, and elsewhere. An RIC summing the intensities of these six ions vs time, shown in Figure 5b, reveals a single peak a t the correct retention time for derivatized 8. An averaged background-subtracted mass spectrum collected under the peak shown in Figure 5b is displayed in Figure 6b. Even though no conspicuous peak for derivatized 8 is apparent in the TIC of derivatized band Z materials (Figure 5a), the RIC in Figure 5b and the agreement of the full spectrum in Figure 6b with that of the standard in Figure 6a confirm that 8, too, is indeed present in the hydrolysate of liver protein from BB-treated rats. A search over the entire G C M S chromatogram of derivatized band Z (i.e., from 6 to 30 min)
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Bromine-containing molecular and fragment ions are conspicuous as 1:l mass doublets (79Brand 81Br)at mlz 235, 237, 360, 362, 461, and 463. To provide for more specific detection of 7 among a high background of nonrelated material, we selected these six ions as a e35
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Chem. Res. Toxicol., Vol. 8, No. 5, 1995 733
BB-3,4-oxideAlkylates Protein N-Nucleophiles
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We propose that 7 and 8, like 6, are formed when metabolically-generated bromobenzene 3,4-oxide alkylates nucleophilic sites in proteins and the initial ringopened adduct is dehydrated and released as shown in Scheme 1. The overall amounts of 7 and 8 are apparently even lower than that of 6, and collectively these three adducts account for only a small part (52.5%)of the total covalent binding of a hepatotoxic dose of P4C1BB to rat liver protein in vivo. We cannot exclude the possibility that ortho andlor meta isomers of 7 and 8 are formed and that for lack of standards we did not detect them. However, we do not believe these isomers are likely to be major components of the unidentified adduct material. First, they would undoubtedly have chromatographed similarly to 6-8 on both XAD-2 and reverse-phase LC. Second, there is little apparent C-3 vs C-4 selectivity in the (enzymatic) conjugation of 1 with glutathione, unlike the case with 2, where C-3 addition is greatly favored over C-2 addition because of steric considerations (26). There could be several reasons that 6-8 are formed in such low amounts in vivo. One is that epoxide 1 is simply not very reactive as an electrophile, leaving spontaneous isomerization to p-bromophenol,enzymatic hydration to a dihydrodiol, and enzyme-catalyzed conjugation with glutathione as the most important contributors to its disposition. Indeed, there is ample evidence to suggest that this is the case (27-30). Although lysine is an abundant amino acid in many proteins and is often located accessibly on their surfaces, many of these side chains are protonated at physiological pH and thus seemingly not available for alkylation. Nevertheless, as noted above, reactive chemicals and metabolites often modify protein lysine side chains under physiologically relevant conditions. Histidine and cysteine (disulfide forms excluded) are of lower overall abundance in pro-
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734 Chem. Res. Toxicol., Vol. 8, No. 5, 1995
teins and are often located more internally rather than exposed at the surface. An important exception,however, would be at the active sites of various enzymes, and this could be of critical importance vis-a-vis the toxicological consequences of protein covalent binding. If the three most nucleophilic amino acids in proteins are only just detectably alkylated by epoxide metabolites of BB, other amino acids are even less likely to be involved. Benzo[a]pyrene diol epoxide metabolites alkylate carboxylate side chains in human serum albumin and hemoglobin, but the adducts are esters and decompose easily to extractable tetrols (31, 32). In contrast, only a small fraction of ['*CIBB-derived radioactivity bound to rat liver protein is extractable after acid hydrolysis, and none of this consists of bromophenols. With so little of the net covalent binding of BB metabolites apparently originating via epoxide metabolites, one must consider that quinone metabolites play a quantitatively significant role in binding. Numerous studies with metabolic inhibitors (29,331,dual-labeled [3W14ClBB (24, 34, 351, and administration o f phenols (36-38) have supported a role for quinone metabolites in covalent binding of bromobenzene. On the other hand, 1,4-benzoquinone, a known metabolite of BB (39),shows negligible reactivity to non-sulfur nucleophiles in the model protein ribonuclease A (40). Quinone-nucleophile adducts are often susceptible to air oxidation and further decomposition leading to complex product mixtures (411, and the broad distribution of I4C during fractionation of hydrolysates of liver protein from BB-treated rats (viz. Figures 1 and 2a) is consistent with this. Methods other than acid hydrolysis should be evaluated for releasing BBprotein adducts for isolation and identification as modified amino acids. Finally, it should be borne in mind that although BB-epoxide adducts to rat liver proteins appear to be minor contributors to net covalent binding in a quantitative sense, it remains possible that they are of major importance toxicologically (20)and that the identity and cellular function of the proteins which become modified could be at least as important as the type of electrophilic metabolite.
Acknowledgment. We thank Dr. Todd Williams for advice and assistance with the mass spectrometry aspects of this research, and the NIH for financial support (GM21784).
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