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Covalent Protein Adducts of Hydroquinone in Tissues from Rats: Identification and Quantitation of Sulfhydryl-Bound Forms† Rodney J. Boatman,* J. Caroline English, Louise G. Perry, and Laurie A. Fiorica Health and Environment Laboratories, Eastman Kodak Company, Rochester, New York 14652-6272 Received February 17, 2000
The Michael-type addition of sulfhydryl groups to benzoquinone (BQ) or substituted benzoquinones is proposed as the primary mechanism by which these electrophilic intermediates react with either cellular glutathione or protein sulfhydryls. This reaction constitutes a reductive alkylation with a substituted hydroquinone (HQ) derivative resulting from the addition. In the case of HQ, oxidative conversion of the parent material to BQ followed by conjugation with glutathione leads to metabolic activation, producing intermediates which are nephrotoxic as well as having other proposed biological activities. Chemically, BQ may react with more than 1 equiv of glutathione (or other sulfhydryl reagents) to produce HQ derivatives substituted with up to four sulfhydryl groups. Similarly, multiply substituted protein-S adducts of HQ were anticipated to occur in vivo following administration of this material. In the current studies, sulfhydryl-bound HQ protein adducts were detected and quantitated in protein isolated from rats using a modification of the alkaline permethylation procedure of Slaughter and Hanzlik [(1993) Anal. Biochem. 208, 288-295]. In particular, total protein-S adducts to HQ in kidney or blood reached a level of 420 or 80 pmol/mg of protein, respectively, 6 h following a single gavage dose of 100 mg/kg HQ. Measured half-lives of protein-S adducts in kidney and blood were 23.9 and 36.0 h, respectively. The applicability of protein-S adducts as a tissue dosimeter for HQ is discussed.
Introduction A growing body of experimental evidence suggests that the conversion of chemicals such as p-aminophenol, hydroquinone (HQ),1 and 2-bromo-HQ to their respective glutathione S conjugates is an obligatory first step in the bioactivation of these materials to nephrotoxic metabolites (1). In the case of hydroquinone, initial oxidation to BQ and subsequent reaction with reduced glutathione produce a monosubstituted conjugate which, under in vivo conditions, may be further oxidized and conjugated with additional glutathione. Sequential oxidation followed by conjugation may yield hydroquinone substituted with up to 4 equiv of glutathione (see Figure 1) (2). In confirmation of this mechanism, Hill et al. (3) have identified the mono-, di-, and tri(glutathion-S-yl)hydroquinone conjugates in bile from male Sprague-Dawley (SD) rats treated ip with HQ (1.8 mmol/kg) and pretreated with AT-125 to inhibit γ-glutamyl transpeptidase (γ-GT) activity. When administered iv to SD rats, all the glutathione conjugates (with the exception of the tetrasubstituted conjugate which was inactive) induced renal toxicity similar to that observed with the parent material but at considerably lower dose levels (2). In this series, 2,3,5-(triglutathion-S-yl)HQ [2,3,5-(triGSyl)HQ] was the most active nephrotoxicant. In addition, Hill et al. (4) † Presented in part at the Society of Toxicology meeting, Dallas, TX, March 13-17, 1994. * Address all correspondence to this author. Phone: (716) 588-5961. Fax: (716) 722-7561. 1 Abbreviations: HQ, hydroquinone; BQ, benzoquinone; 2,3,5-(triGSyl)HQ, 2,3,5-(triglutathion-S-yl)hydroquinone; TCA, trichloroacetic acid.
have recently reported that radiolabeled 2,3,5-(triGSyl)HQ was more strongly retained by the in situ perfused rat kidney than was the monosubstituted homologue, suggesting that the more highly substituted conjugate or metabolites derived from it are more reactive and thus potentially more cytotoxic. Though still a matter of debate (5), it has been proposed that protein binding of reactive quinone or quinone thioethers is necessary for the cytotoxicity of these materials. By this mechanism, a target protein within a tissue may react with a quinone or a quinone metabolite to form a protein-S adduct completely analogous to the corresponding reaction with reduced glutathione. It has been shown recently by Hanzlik et al. (6) that in the model reaction of ribonuclease A with BQ, covalent binding to protein sulfhydryls occurs exclusively with no significant binding to other nucleophilic protein side chains. These same investigators further suggest that under physiologically relevant conditions (i.e., low concentrations of reactive metabolites and short reaction times), sulfhydryl arylation by BQ would be the only type of reaction observed. Quinone protein-S adducts are sufficiently unstable such that traditional isolation techniques involving hydrolysis followed by amino acid analysis are not suitable (7). Several methods, however, do exist for the detection and/or quantitation of sulfhydryl-bound HQ and substituted HQ derivatives. Thus, the method of McDonald et al. (8), employing Raney nickel reduction of protease hydrolysates of serum and bone marrow protein, generates HQ. This is subsequently derivatized and analyzed by gas chromatography/mass spectrometry (GC/MS) or
10.1021/tx000037x CCC: $19.00 © 2000 American Chemical Society Published on Web 08/11/2000
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Figure 1. (A) Product of the reaction of benzoquinone with reduced glutathione. (B) Substitutions of one, two, three, or four glutathionS-yl residues on the HQ nucleus.
gas chromatography/electron capture detection (GC/ ECD). Although this method has sufficient sensitivity to detect background levels of protein-bound HQ in bone marrow and blood, the reduction reaction itself presumably cleaves all HQ-thiol bonds, thus providing no information concerning the positions or extent of sulfhydryl substitution. More recently, Rombach and Hanzlik (9) have described a polyclonal antibody specific for the thiol-bound S-(2,5-dihydroxyphenyl) moiety. This “antiHQ” antibody exhibits a high specificity for protein-HQ adducts to liver proteins in rats following incubation of bromobenzene with rat liver microsomes. The specificity of this antibody for more highly conjugated forms of HQ, however, is not reported. As an alternative, we have employed the alkaline “permethylation” procedure described originally by Slaughter and Hanzlik (7) and more completely in a subsequent publication by Slaughter et al. (10). In this procedure, basic hydrolysis of tissue proteins results in a chemical fragmentation of sulfhydryl-substituted HQ intermediates which, in the presence of excess methyl iodide, are rapidly converted to the chemically stable methylthio derivatives as shown in Figure 2. A GC/MS procedure was developed which allowed for the simultaneous quantitation of all six of the possible sulfhydryl-substituted HQ products. To calibrate this procedure, the glutathione conjugates of HQ were synthesized as standards and subjected to the permethylation conditions used to derivatize protein samples.
Experimental Procedures Materials. Hydroquinone (HQ) and benzoquinone (BQ) were obtained from the Eastman Kodak Co. (Rochester, NY). The chemical purities of these were determined to be >99% by HPLC. Reduced glutathione (96%) was obtained from the Aldrich Chemical Co. (Milwaukee, WI). Methyl iodide and resorcinol were obtained from the Aldrich Chemical Co. and were reagent grade. Pentane (Aldrich Chemical Co.) was HPLCgrade. Heptane (glass-distilled) was obtained from EM Sciences, Inc. (Cherry Hill, NJ) and was chromatographic grade. All other chemicals were reagent grade unless otherwise noted.
Figure 2. Methylthio-substituted products resulting from the permethylation of either glutathione- or protein-derived sulfhydryl adducts of hydroquinone. Animals. Specific details, including the source and age of animals used in these studies, can be found in the following paper (11). Dose Preparation, Administration, and Analysis. Stock solutions of HQ were prepared in degassed, distilled water or degassed, sterile saline at concentrations not exceeding 50 mg/ mL. Rats were given HQ by oral gavage in degassed, distilled water or by intraperitoneal injection in degassed, sterile saline at one or more dose levels as described in the following paper (11). The concentration of HQ in treatment solutions was determined by HPLC immediately prior to use. Synthesis of HQ Glutathione Conjugates. The glutathione conjugates of hydroquinone were prepared as standards by a modification of the procedure of Lau et al. (2) and purified by preparative HPLC. In a typical procedure, 0.5022 g (4.646 mmol) of BQ was placed in a 50 mL Erlenmeyer flask and dissolved in 10-11 mL of methanol. With stirring and under a continuous nitrogen purge, a solution of 1.2517 g (4.073 mmol) of reduced glutathione in 10-11 mL of distilled, deionized water
Quantitation of HQ Protein Adducts was added dropwise to the BQ solution. A thick precipitate formed which required the addition of 30 mL of nitrogen-purged distilled, deionized water to dissolve. Following a brief (24 h) to produce free-flowing, off-white solids. Samples of whole blood were treated with equal volumes of 40% TCA, and the protein precipitates washed sequentially with acetone (six times), 80% methanol/H2O (four times), acetone (four times), and ethyl ether (four times). Solid blood protein samples were dried initially with a gentle stream of nitrogen followed by application of high vacuum (>24 h) to produce free-flowing solids. Alkaline Permethylation. Methylthio-substituted derivatives of 1,4-dimethoxybenzene, resulting from the alkaline permethylation of tissue, urine, or blood protein, were obtained using a modification of a published procedure (7, 10). In the case of tissue proteins, approximately 125 mg samples were placed into PTFE-lined, screw-capped culture tubes (16 mm × 100 mm) followed by 1.0 mL of methyl iodide. Resorcinol as an internal standard was added at this point (50 µL of a 0.1 mg/g of solution in distilled water containing 0.5 mg/g of ascorbic acid). Dry nitrogen was gently bubbled through the mixture, and 1.0 mL of nitrogen-purged, 4 N NaOH (purged a minimum of 10 min with nitrogen immediately prior to use) was then added. Tubes were flushed for an additional 30 s with nitrogen, capped tightly, and heated in thermostatically controlled heated blocks at 80 °C for 4 h. Following this, the tubes were cooled in ice and opened. A 0.75 mL aliquot of 14 N NaOH was cautiously added to each tube followed by 4 mL of methanol and acetone (1:1,
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Boatman et al. film thickness), 5 psi head pressure (He); oven program, 100 °C initial (hold 3 min), 10 °C/min to 300 °C (hold 5 min); injector at 300 °C, split flow of 12 mL/min; MSD used in a single-ion mode (150 ms dwell times) with ions monitored at m/z 138.1 (3-8 min, resorcinol), 184.1 (8-12 min, mono), 230.0 [12-16 min; di(2,3), di(2,5), and di(2,6)], 276.0 (16-18 min, tri), and 322.1 (18-20 min, tetra). Statistical Analyses. The results for individual and total protein-S adduct levels were analyzed statistically using the computer program SAS (version 6, SAS Institute, Cary, NC).
Results and Discussion
Figure 3. Concentrator apparatus used for the permethylation of protein samples, which was constructed by joining 7 mm (o.d.) glass tubing to 12 mm (o.d.) glass tubing. It was designed to contain e5 mL of liquid when filled. v:v). After being cooled in ice for approximately 5 min, a 4 mL aliquot of pentane was added to each tube, and the tubes were vortically mixed and the phases allowed to separate. The organic pentane extracts were separated. The mixtures were extracted a total of three times with pentane and pooled pentane extracts washed four times with small volumes of water and finally dried over granular, anhydrous sodium sulfate. Initially, the pentane extracts were concentrated by placing them into pear-shaped flasks (50 mL) equipped with small (10-15 cm in length) Vigreux columns and heating these in a 50 °C water bath to evaporate the majority of solvent. When the volume had reduced to approximately 0.5-1 mL, the residues were transferred into specially designed concentrator units as shown in Figure 3. A small boiling chip was added, and the end of each concentrator was immersed in a 50 °C water bath. The solvent was allowed to evaporate from each unit until all residual pentane had completely disappeared, at which point the concentrators were removed from the water bath. A 20 µL aliquot of n-heptane was added to each concentrator, and the concentrators were returned to the heated bath for an additional 1 h to ensure removal of all residual pentane. The concentrators were then scored and broken as indicated in Figure 3 and the residues (20-50 µL) analyzed by GC/MS. Concentrated, stock solutions of the glutathione conjugates of HQ (prepared in degassed, distilled water containing 0.5% ascorbic acid to inhibit oxidation) were added to tubes (by weight) and subjected to identical permethylation conditions. GC/MS Analysis Conditions. Quantitative determinations of the various methylthio-substituted derivatives were made by comparisons of integrated ion intensities from sample analyses (single-ion mode) to the results obtained by permethylation of synthetically prepared HQ glutathione conjugates used as standards (resorcinol as an internal standard). Calibration standards were prepared over the range necessary to encompass levels observed in permethylated tissue protein (1-50 nmol/ sample). Separate calibration curves for individual permethylated components (Figure 2) were obtained in which relative area responses (ratio of calibration standard to internal standard) were plotted versus amount ratios. The calibration curves obtained were linear over the ranges analyzed with correlation coefficients (r2) generally exceeding 0.98. Insufficient amounts of pure 2,3-(diglutathion-S-yl)hydroquinone were obtained to use as a GC/MS standard. In this latter case, the calibrated response for the permethylated 2,5-(diglutathion-S-yl)hydroquinone standard was used to quantitate amounts of the 2,3-isomer. The following chromatographic conditions were employed for these analyses: instrument, Hewlett-Packard HP 5890 gas chromatograph with a HP 5970 mass selective detector (MSD); column, DB-5 (J & W Scientific, Folsom, CA), 30 m × 0.25 mm (0.1 µm
BQ and other oxidized metabolites of HQ containing the quinone moiety are capable of reacting with tissue protein, producing protein-S-bound adducts. Thus, covalently bound HQ (as well as bromo-HQ) has been identified in liver protein from rats treated with bromobenzene (7), in hemoglobin and bone marrow protein from rats treated with benzene (13), and has been tentatively identified in hemoglobin and liver protein from mice treated with acetaminophen (14). In a recent publication, Hill et al. (4) demonstrated that protein binding occurs within the in situ perfused rat kidney following administration of the nephrotoxic glutathione conjugates of HQ. In these studies, the extents of binding of radiolabeled substrates and excretion of the brush border membrane-bound enzyme, γ-GT, were greatest for the triglutathionyl conjugate, 2,3,5-(triglutathion-S-yl)hydroquinone. The goal of the current studies was to determine the extent and nature of the covalent binding of HQ in rats following either oral or ip administration and to determine the relevance of this tissue dosimeter to the known acute and chronic toxicities of HQ. Identification and Quantitation of HQ Thiol Adducts to Rat Liver Protein. As shown in Figure 4, all six of the methylthiol-substituted adducts of HQ were identified by GC/MS in liver and kidney proteins from a male F344 rat treated ip with HQ at 100 mg/kg (11). The specific structural assignments for all of the substituted methylthiol derivatives were determined by retention time and mass spectral comparisons to the corresponding permethylated glutathionyl-substituted standards. Mass spectra of permethylated components from tissue samples were nearly identical with spectra derived from permethylated standards. In all cases, the total ion mass spectra of these methylthiol derivatives indicated that the molecular (M+) ions are the most abundant ions in the spectra. Fragmentation with a successive loss of CH3 groups leads to the observed spectra containing significant (M - 15)+, (M - 30)+, and, in some cases, (M - 45)+ ions. This distinctive fragmentation pattern, characterized by successive loss of methyl groups, was common to all the methylthiol derivatives. As further confirmation of these structural assignments, the contribution of the 34S isotope to the intensity of the (M + 2)+ ions in these spectra was clearly seen to increase from approximately 5% in the mono derivative to 20% in the tetra derivative. Calibration curves generated from permethylated standards allowed accurate quantitation of individual methylthio derivatives in the range of 1-50 pmol/sample (based on a 125 mg protein sample). Stability Study. The in vivo stability of the proteinbound forms of hydroquinone was determined in male F344 rats. Tissues chosen for examination in this study included blood and kidney. Both total protein-S adduct levels as well as individual adduct levels were measured
Quantitation of HQ Protein Adducts
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 857 Table 2. Levels of Individual HQ Protein-S Adducts versus Time of Collection for Blood Protein from Groups of Five Male F344 Rats Treated Orally with HQ (100 mg/kg)a adduct level (pmol/mg of protein) 6h 24 h 48 h
individual adduct
control
mono 2,3-di 2,5-di 2,6-di tri tetra
6.30 ( 1.99 NDb ND 1.69 ( 0.757 ND ND
a
38.0 ( 5.70 6.55 ( 0.977 13.1 ( 1.66 13.3 ( 3.79 8.91 ( 1.19 ND
21.9 ( 4.08 5.02 ( 0.874 8.14 ( 1.42 6.67 ( 1.04 6.07 ( 0.847 ND
16.0 ( 3.31 3.74 ( 2.13 4.96 ( 2.80 5.25 ( 0.666 5.14 ( 0.755 ND
Error expressed as means ( 1 SD. b ND, none detected.
Table 3. Levels of Individual HQ Protein-S Adducts versus Time of Collection for Kidney Protein from Groups of Five Male F344 Rats Treated Orally with HQ (100 mg/kg)a individual adduct mono 2,3-di 2,5-di 2,6-di tri tetra a
Figure 4. GC/MS chromatographic profiles for the methylthiolsubstituted HQ derivatives: 2-methylthio-1,4-dimethoxybenzene, retention time of 10.5 min; 2,3-bis(methylthio)-1,4dimethoxybenzene, retention time of 14.2 min; 2,5-bis(methylthio)-1,4-dimethoxybenzene, retention time of 14.3 min; 2,6-bis(methylthio)-1,4-dimethoxybenzene, retention time of 14.8 min; 2,3,5-tris(methylthio)-1,4-dimethoxybenzene, retention time of 17.5 min; and 2,3,5,6-tetrakis(methylthio)-1,4-dimethoxybenzene, retention time of 18.9 min. (A) Representative chromatogram of permethylated glutathionyl-HQ standards. (B) Permethylated liver protein from a male F344 rat treated ip with HQ at 100 mg/kg (11). (C) Permethylated kidney protein from a male F344 rat treated ip with HQ at 100 mg/kg (11). Table 1. Levels of Total Protein-S Adducts in Blood and Kidneys from Groups of Five Male F344 Rats before Treatment (0 h) and after a Single Oral Dose of HQ (100 mg/kg)a total protein-S adduct level (pmol/mg of protein) collection time (h)
blood
kidney
0 (control) 6 24 48
7.06 ( 79.9 ( 11.0 47.8 ( 7.88 35.0 ( 9.38
14.2 ( 4.19 420 ( 51.9 255 ( 41.3 124 ( 42.8
3.44b
a Tissues from groups of rats were collected before treatment (0 h) and 6, 24, and 48 h after treatment. b Results are expressed as means ( 1 SD.
in blood and kidney proteins obtained from groups of rats (n ) 5) sacrificed at 6, 24, or 48 h following a single gavage administration of 100 mg/kg HQ. Total protein-S adduct levels found in the tissues at the various collection times are shown in Table 1. Individual adduct concentrations (picomoles per milligram of protein) versus time of
adduct level (pmol/mg of protein) control 6h 24 h 48 h 5.24 ( 0.943 3.03 ( 2.82 NDb 0.691 ( 0.661 2.71 ( 0.577 2.53 ( 2.58
76.7 ( 7.03 18.5 ( 3.41 28.3 ( 2.45 28.6 ( 3.69 85.0 ( 9.48 183 ( 33.2
42.6 ( 7.81 12.6 ( 2.62 9.91 ( 1.12 13.4 ( 1.58 57.2 ( 7.09 120 ( 25.8
21.2 ( 6.85 7.15 ( 1.98 1.77 ( 2.60 5.31 ( 4.45 30.2 ( 9.54 58.7 ( 19.4
Error expressed as means ( 1 SD. b ND, none detected.
sampling are shown for blood in Table 2 and for kidney in Table 3. In either tissue protein, adduct levels (both total and individual) were found to decrease steadily after 6 h, with calculated, mean half-lives of 23.9 h for kidney and 36.0 h for blood protein-S adducts. Decreases in HQ protein-S adduct levels within a tissue are determined not only by the chemical stability of the initially formed adducts but also by the inherent protein turnover rate for the particular tissue. In this regard, adduct stability in protein from whole blood (primarily hemoglobin) versus that from kidney provides a useful comparison for these two dissimilar tissues. In particular, the turnover rate or lifespan of hemoglobin (Hb) is unique in that it is equivalent to the lifespan of the erythrocyte, which for the rat is approximately 60 days (15). Similarly, half-lives for chemically stable hemoglobin adducts are generally long, with the lifetimes of the adducts determined primarily by that of the erythrocyte life span (15, 16). The relatively short (36 h) half-life of HQ blood protein-S adducts in this study suggests that under in vivo conditions these adducts are not stable but undergo further oxidative transformations and are, as a consequence, no longer detected by permethylation. Oxidation within the red blood cell by hemoglobin itself provides a likely explanation for this decreased stability. In this regard, hemoglobin has been shown to display both monooxygenase-like and peroxygenase-like activities toward carcinogenic primary arylamines (17). On the basis of the criteria of Skipper and Tannenbaum (16), the relatively short half-life of the blood protein-S adducts of HQ makes them a less useful (sensitive) tissue dosimeter for total HQ exposure. The fate of HQ protein-S adducts in the kidney provides a significant contrast to that in blood. Although adducts to kidney protein have a half-life similar to that measured in blood (24 vs 36 h), the rate of protein turnover within this tissue is far greater than that of hemoglobin, and this more rapid turnover provides an explanation for the half-life of the HQ kidney protein-S
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Figure 5. Proposed scheme for the formation of multiply substituted glutathione- and protein-sulfhydryl adducts of HQ.
adducts seen in this study. Thus, Goldspink and Kelly (18) report relatively consistent half-life values for mixed kidney proteins from male CD rats ranging from 2 days (at 8 weeks of age) to 3 days (at 105 weeks of age). Also, it can be anticipated that the rate of protein turnover within the metabolically active proximal tubular epithelial cells will be similar to or greater than that for mixed kidney protein. In support of this, Tsao et al. (19) report an in vitro half-life of 32 h (turnover rate of 2.18%/h) for protein in freshly isolated proximal tubules from male SD rats. Thus, results from the current study are consistent with protein turnover being the primary
determinant of the measured half-life of HQ kidney protein-S adducts in the rat. The permethylation procedure used in the current studies provides a rapid and sensitive method for the quantitation of quinone-derived protein thiol adducts. However, the degradative nature of the derivitization precludes the possibility of direct identification of the protein precursors. Thus, for methylthio-HQ derivatives containing two or more methylthio groups, it is not possible to know which or how many of the methylthio groups derive from a protein sulfhydryl in the initial adduct. Also, the extent of intra- and interprotein cross-
Quantitation of HQ Protein Adducts
Chem. Res. Toxicol., Vol. 13, No. 9, 2000 859
Table 4. Protein-S-Bound Forms of HQ Expressed as a Percentage of the Administered Dosea protein-S-bound HQ expressed as a percentage of the administered dose male F344 rats
female F344 rats
study (dose)
liver
kidney
liver
kidney
currentb (25 mg/kg) [14C]HQ, oralc (25 mg/kg)
0.25 0.075
0.015 0.010
0.39 0.23
0.031 0.020
a Values from the current study are compared with residual tissue radioactivity in rats treated orally with [14C]HQ. b See ref 11. c Male and female F344 rats, 7-9 weeks of age, were treated orally with [14C]HQ (25 mg/kg). In the case of male rats, tissues were collected 72 h after treatment. For females, tissues were collected 48 h after treatment.
linking cannot be determined by the procedure described. As a simplifying assumption, it is proposed that the majority of multiply S-substituted methylthio derivatives of HQ identified in the current studies are derived from protein precursors in which the HQ moiety is linked through a single protein thiol group. This assumption is based on the fact that oxidized quinone precursors will preferentially react with glutathione, which is the predominant thiol-containing material in nearly all mammalian cells, present at millimolar concentrations (20), and consequently, reaction with protein sulfhydryls will be far less favored. Additional support for this contention comes from the work of Hanzlik et al. (6), who report little or no intermolecular cross-linking of ribonuclease in vitro after treatment with BQ at levels as high as 64 mol of BQ/mol of protein. A simplified scheme for the formation of multiply S-substituted protein sulfhydryl adducts of HQ is shown in Figure 5. The protein-S-bound adducts identified in the current studies represent less than 1% of the administered dose in the liver and less than 0.1% of the administered dose in the kidneys. Using published values (21-23) for the protein contents of liver and kidneys of rats (milligrams of protein per gram of tissue wet weight), adduct concentrations (picomoles per milligram of protein) were converted to total adduct equivalents in the respective tissues. In the case of liver, the highest adduct levels that were obtained corresponded to 0.89% and 0.54% of the administered doses following repeat oral and single ip administrations, respectively (11). The corresponding highest values for kidney adducts were 0.07% of the administered dose following repeat oral administration and 0.09% of the administered dose following single ip administration. Thus, protein arylation by HQ and reactive HQ metabolites represents only a small percentage of the administered dose of HQ. It has been previously suggested that under physiologically relevant conditions, sulfhydryl-bound forms of HQ bound to protein should predominate (6). In Table 4, protein-S adducts in livers and kidneys of male and female F344 rats determined in the current studies (expressed as a percentage of the administered dose) are compared with the residual radioactivity in tissues from animals treated orally with [14C]HQ (unpublished results from this laboratory). Although these values are uncorrected for protein turnover, the agreement is remarkably good and provides convincing evidence that the sulfhydryl-bound forms of HQ are, in fact, the primary adducts present in these tissues.
Conclusions Sulfhydryl-bound forms of HQ were found in tissue proteins from rats following either gavage or ip administration. Permethylation allowed identification and quantitation of all six of the possible protein-S-bound forms of HQ in which the HQ nucleus is substituted with one, two, three, or four methylthio substituents. In addition, this work provides the first conclusive evidence for the existence of the more highly substituted protein-S-bound forms of HQ (containing either three or four methylthio substituents). The following paper (11) will explore protein-S-bound adduct levels in tissues of rats following either single gavage or ip administration of HQ or following 6 weeks of repeated gavage administrations of HQ (11).
Acknowledgment. We are grateful for the advice and suggestions provided by Prof. Robert P. Hanzlik of the University of Kansas (Lawrence, KS). Financial support was provided by Eastman Chemical Co., Mitsui Petrochemicals (America), Ltd., and Rhodia, Inc.
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