Identification of Quinol Thioethers in Bone Marrow of Hydroquinone

Shawn B. Bratton, Serrine S. Lau, and Terrence J. Monks ... Judy L. Bolton, Michael A. Trush, Trevor M. Penning, Glenn Dryhurst, and Terrence J. Monks...
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Chem. Res. Toxicol. 1997, 10, 859-865

859

Identification of Quinol Thioethers in Bone Marrow of Hydroquinone/Phenol-Treated Rats and Mice and Their Potential Role in Benzene-Mediated Hematotoxicity Shawn B. Bratton, Serrine S. Lau, and Terrence J. Monks* Division of Pharmacology and Toxicology, College of Pharmacy, University of Texas at Austin, Austin, Texas 78712 Received December 19, 1996X

Metabolism of benzene is required to produce the classical hematological disorders associated with its exposure. After coadministration of hydroquinone (0.9 mmol/kg, ip) and phenol (1.1 mmol/kg, ip) to male Sprague-Dawley rats and DBA/2 mice, 2-(glutathion-S-yl)hydroquinone was identified in the bone marrow of both species. 2,5-Bis(glutathion-S-yl)hydroquinone, 2,6bis(glutathion-S-yl)hydroquinone, and 2,3,5-tris(glutathion-S-yl)hydroquinone were also observed in the bone marrow of rats but were detected only sporadically in mice. Both species produced 2-(cystein-S-ylglycinyl)hydroquinone, 2-(cystein-S-yl)hydroquinone, and 2-(N-acetylcystein-S-yl)hydroquinone, indicating the presence of a functional mercapturic acid pathway in bone marrow. The ability of bone marrow to acetylate 2-(cystein-S-yl)hydroquinone and deacetylate 2-(N-acetylcystein-S-yl)hydroquinone was confirmed in vitro. Total quinol thioether concentrations were higher in, and eliminated more slowly from, the bone marrow of mice. Intravenous injection of 100 µmol/kg 2-(glutathion-S-yl)hydroquinone to rats gave rise to substantially lower bone marrow Cmax and AUC values compared to values found following coadministration of hydroquinone/phenol, suggesting that the major fraction of the GSH conjugates present in bone marrow are formed in situ. Finally, the erythrotoxicity of several of these conjugates was determined in rats using the erythrocyte 59Fe incorporation assay. Administration of 2,3,5-tris(glutathion-S-yl)hydroquinone (17 µmol/kg, iv), 2,6-bis(glutathionS-yl)hydroquinone (50 µmol/kg, iv), and benzene (11 mmol/kg, sc) significantly decreased 59Fe incorporation into reticulocytes to 45 ( 6%, 28 ( 3%, and 20 ( 9% of control values, respectively. Although the doses of 2,3,5-tris(glutathion-S-yl)hydroquinone and 2,6-bis(glutathion-S-yl)hydroquinone represented only 0.2% and 0.4% of the dose of benzene, both conjugates reduced 59Fe incorporation to the same degree as benzene. 2-(Glutathion-S-yl)hydroquinone had no effect at the dose tested (200 µmol/kg, iv). In summary, these data suggest that hydroquinoneglutathione conjugates are erythrotoxic and may contribute to benzene-mediated hematotoxicity.

Introduction Benzene is an important industrial chemical, a component of gasoline, and an environmental pollutant (1, 2). Human exposure to benzene has led to a variety of hematological disorders, including leukopenia, anemia, thrombocytopenia, pancytopenia, aplastic anemia, and leukemia (3-5). While no good animal model exists to study benzene’s leukemogenic effects, its hematotoxic effects have been reproduced in several species (6, 7). Biotransformation of benzene is an absolute requirement for toxicity (8-10), and a number of studies with rats and mice indicate that species- and strain-dependent differences in sensitivity to benzene poisoning are often attributable to differences in metabolism (11, 12). Hepatic cytochromes P450 convert benzene to various phenolic metabolites, including phenol, hydroquinone, catechol, resorcinol, 1,2,4-benzenetriol, and the ringopened metabolites trans,trans-muconaldehyde and trans,trans-muconic acid (13-15). Although none of these metabolites are capable of reproducing the myelotoxic effects of benzene on an individual basis (16), a combination of metabolites has proven successful. In particular, Eastmond et al. (17) have shown that the * Address correspondence to this author at the above address. Tel: (512) 471-6699. Fax: (512) 471-5002. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, July 15, 1997.

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combination of hydroquinone (0.23-0.68 mmol/kg) and phenol (0.8 mmol/kg) decreases total bone marrow cellularity in mice in a dose-dependent manner. Other investigators have shown that coadministration of hydroquinone and phenol to mice damages bone marrow pronormoblasts and normoblasts (using the 59Fe incorporation assay) (18) and increases the formation of micronuclei in polychromatic erythrocytes (19), changes consistent with erythroid cytotoxicity and genotoxicity, respectively. The effectiveness of this combination appears to be related to a pharmacokinetic interaction between hydroquinone and phenol, which leads to significantly higher levels of both metabolites in bone marrow (20). Phenol likely competes with hydroquinone for conjugative enzymes and/or depletes the liver of uridine diphosphate glucuronic acid and/or phosphoadenosine phosphosulfate, resulting in greater fractions of hydroquinone and phenol available for delivery to bone marrow (20). Benzene-induced bone marrow suppression has been suggested to result from peroxidase and/or phenoxy radical-mediated oxidation of hydroquinone, leading to the reactive intermediates 1,4-benzosemiquinone and 1,4-benzoquinone. These metabolites may either directly arylate tissue macromolecules and/or redox cycle with the concomitant formation of reactive oxygen species (21-25). © 1997 American Chemical Society

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1,4-Benzoquinone also undergoes nucleophilic addition with GSH,1 leading to the formation of various GSH conjugates, including 2-(glutathion-S-yl)hydroquinone [2-(GSyl)HQ], 2,5-bis(glutathion-S-yl)hydroquinone [2,5bis(GSyl)HQ], 2,6-bis(glutathion-S-yl)hydroquinone [2,6bis(GSyl)HQ], and 2,3,5-tris(glutathion-S-yl)hydroquinone [2,3,5-tris(GSyl)HQ] (26). When administered to rats, each of these conjugates is nephrotoxic in a manner dependent upon the activity of γ-glutamyl transpeptidase (27). Because the redox properties of these conjugates and their metabolites indicate they are more chemically (re)active than hydroquinone (26, 28, 29), we sought to determine whether these quinol thioethers are present in the bone marrow of rats and mice following coadministration of hydroquinone and phenol, or benzene, and whether they have the potential to contribute to the hematotoxicity observed in this model of benzene toxicity.

Materials and Methods Caution: Hydroquinone is a potential carcinogen and must be handled accordingly. In addition, the synthetic thioethers are potent nephrotoxicants in the rat. All these compounds should therefore be handled with protective clothing in a well-ventilated fume hood. Materials. 2-(GSyl)HQ, 2,5-bis(GSyl)HQ, 2,6-bis(GSyl)HQ, 2,3,5-tris(GSyl)HQ, 2-(cystein-S-ylglycine)hydroquinone [2-(CysGly)HQ], 2-(cystein-S-yl)hydroquinone [2-(Cys)HQ], and 2-(Nacetylcystein-S-yl)hydroquinone [2-(NAC)HQ] were all synthesized according to previously published methodologies (26). Hydroquinone was purchased from Fluka Chemika (Buchs SG, Switzerland) and phenol from EM Science (Gibbstown, NJ). GSH, ascorbic acid, CoASAc, EDTA, ammonium acetate, and acivicin were obtained from Sigma Chemical Co. (St. Louis, MO). Citric acid (anhydrous) and HPLC-grade methanol were purchased from Fisher Scientific (Fair Lawn, NJ). N-Acetyl-Lcysteine and perchloric acid were obtained from the Aldrich Chemical Co. (Milwaukee, WI). 59FeCl3 (8.76 mCi/mg, 99% pure) was purchased from DuPont NEN Life Science Products (Boston, MA). Animals. Male Sprague-Dawley rats (175-200 g) and DBA/2 mice (20-25 g) were purchased from Harlan SpragueDawley (Houston, TX) and used for all metabolism and 59Fe uptake experiments. All animals were housed on a 12 h light/ dark cycle and allowed food and water ad libitum. Treatment Protocols and Isolation of Bone Marrow. Male Sprague-Dawley rats and DBA/2 mice were either administered benzene (11.2 mmol/kg, sc in corn oil) twice daily for 2 days or coadministered hydroquinone (0.9 mmol/kg, ip) and phenol (1.1 mmol/kg, ip) dissolved in a vehicle consisting of 0.85% phosphate-buffered saline (91%):ethanol (9%). Controls received only the vehicle. At various time points after injection (1, 5, 10, 15, 30, and 60 min for hydroquinone/phenol and 2 h for benzene) animals were euthanized by cervical dislocation, and each femur was quickly removed, cleansed of muscle, and placed on ice. The epiphyseal plates of each femur were then removed, and the marrow was flushed with 1 mL (rats) or 0.5 mL (mice) of an ice-cold solution of 0.1 N perchloric acid containing 130 mM EDTA and 260 mM Na2S2O5. The cell suspensions were quickly probe-sonicated for 20 s and placed on ice. A 200 µL aliquot of the homogenate was withdrawn for protein analysis (Bradford), and the remaining sample was centrifuged (Eppendorf, Model 5415c) for 10 min at 13 500 rpm, chilled, and recentrifuged for an additional 10 min. The clear 1 Abbreviations: 2-(GSyl)HQ, 2-(glutathion-S-yl)hydroquinone; 2,5bis(GSyl)HQ, 2,5-bis(glutathion-S-yl)hydroquinone; 2,6-bis(GSyl)HQ, 2,6-bis(glutathion-S-yl)hydroquinone; 2,3,5-tris(GSyl)HQ, 2,3,5-tris(glutathion-S-yl)hydroquinone; 2-(Cys-Gly)HQ, 2-(cystein-S-ylglycinyl)hydroquinone; 2-(Cys)HQ, 2-(cystein-S-yl)hydroquinone; 2-(NAC)HQ, 2-(N-acetylcystein-S-yl)hydroquinone; CoASAc, acetyl coenzyme A; AUC, area under the bone marrow concentration-time curve.

Bratton et al. supernatant was then removed and 20 µL used for HPLC analysis. The remaining sample was stored at -80 °C. Determination of Microsomal Cysteine Conjugate NAcetyltransferase and Cytosolic N-Acetylcysteine Conjugate N-Deacetylase Activities. Enzyme assays were conducted according to the method of Lau et al. (30) with minor modifications. Briefly, bone marrow was flushed from each femur with 1 mL of ice-cold Tris-KCl buffer (pH 7.4; 20 mM Tris, 0.15 M KCl). The tissue was homogenized with a glass homogenizer, and the microsomal/cytosolic fractions were prepared by standard differential centrifugation at 4 °C at 100000g. Assays of cysteine conjugate N-acetyltransferase activity contained 0.9 mM ascorbic acid, 90 µM 2-(Cys)HQ (substrate was never rate-limiting), either rat (152 µg of protein) or mouse (3.6 µg of protein) bone marrow microsomes, and 360 µM CoASAc in 0.1 M potassium phosphate buffer (pH 7.4) (550 µL final volume). Mixtures were preincubated for 2 min at 37 °C, and reactions were initiated with the addition of CoASAc. Reactions were terminated with 10% perchloric acid (50 µL) at 20 and 30 min for rats and mice, respectively. Similarly, assays of N-acetylcysteine conjugate N-deacetylase activity consisted of 1 mM ascorbic acid, either rat (240 µg) or mouse (6.4 µg) bone marrow cytosol, and 100 µM 2-(NAC)HQ (substrate was never rate-limiting) in 0.1 M phosphate buffer (pH 7.4) (500 µL final volume). Mixtures were preincubated for 2 min at 37 °C, and reactions were initiated with the addition of the substrate 2-(NAC)HQ. The reactions were terminated with 10% perchloric acid (50 µL) at 40 and 60 min for rats and mice, respectively. All samples were processed and stored as described above. Uptake of 2-(GSyl)HQ from the Circulation into Bone Marrow. Male Sprague-Dawley rats were administered 100 µmol/kg 2-(GSyl)HQ dissolved in 0.85% phosphate-buffered saline via tail vein injection. At various time points (1, 2.5, 5, 10, and 30 min), the rats were euthanized by cervical dislocation (acccording to IACUC guidelines), and bone marrow samples were obtained and analyzed as described above. The total amount of quinol thioethers excreted in urine and bile following administration of hydroquinone (1.8 mmol/kg, ip) represents about 4.3% of the dose (28). Therefore the dose of 2-(GSyl)HQ (100 µmol/kg, iv) administered to rats in this experiment almost certainly exceeds the total peripheral conversion of hydroquinone/phenol (2.0 mmol/kg, ip) to GSH conjugates of hydroquinone, especially since 100% conversion of phenol (1.1 mmol) to hydroquinone is highly unlikely. HPLC Analysis of Quinol Thioethers. Bone marrow concentrations of all metabolites were determined by HPLC coupled to an eight-channel coulometric electrode array system (HPLC-CEAS; ESA Inc., Chelmsford, MA). Electrode potentials were increased from 0 to 350 mV in 50 mV increments. The mobile phase consisted of 4 mM citrate, 8 mM ammonium acetate, 20 mg/L EDTA, and methanol (0-2% over 50 min), pH 4.0. The flow rate was kept constant at 0.6 mL/min, and separation was achieved using a Partisil 5 ODS-3 reverse-phase analytical cartridge column (4.6 mm × 25 cm) (Whatman, Clifton, NJ). Under these conditions, the retention times (min) of authentic standards were as follows: hydroquinone (12.7), 2-(GSyl)HQ (20.8), 2,5-bis(GSyl)HQ (34.6), 2,6-bis(GSyl)HQ (39.3), 2,3,5-tris(GSyl)HQ (23.3), 2-(Cys-Gly)HQ (17.7), 2-(Cys)HQ (14.6), and 2-(NAC)HQ (27.2). Quantitation of each metabolite was determined by comparing its peak area with its corresponding standard curve, prepared between 4 and 200 pmol for 2,5-bis(GSyl)HQ, 2,6-bis(GSyl)HQ, and 2,3,5-tris(GSyl)HQ and between 0.4 and 200 pmol for 2-(GSyl)HQ, 2-(Cys-Gly)HQ, 2-(Cys)HQ, and 2-(NAC)HQ. Hematotoxic Potential of Quinol Thioethers Determined by the 59Fe Incorporation Assay. Benzene toxicity to (pro)normoblasts in bone marrow subsequently reduces the size of the reticulocyte pool and thus the requirement for iron for hemoglobin synthesis. To determine the potential adverse effects of the GSH conjugates on (pro)normoblasts, we utilized the 59Fe incorporation assay described by Lee et al. (31, 32) with slight modifications. Briefly, male Sprague-Dawley rats were

Quinol Thioether-Mediated Hematotoxicity

Figure 1. Identification and quantitation of quinol thioether metabolites in rat bone marrow. Following coadministration of hydroquinone (0.9 mmol/kg, ip) and phenol (1.1 mmol/kg, ip), hydroquinone-glutathione conjugates (A) and mercapturic acid pathway metabolites (B) were quantified in rat bone marrow by HPLC-CEAS: (A) (O) hydroquinone, (b) 2-(glutathion-S-yl)hydroquinone, (O, dashed line) 2,5-bis(glutathion-S-yl)hydroquinone, (0) 2,6-bis(glutathion-S-yl)hydroquinone, (9) 2,3,5tris(glutathion-S-yl)hydroquinone; (B) (b) 2-(glutathion-Syl)hydroquinone, (0, dashed line) 2-(cystein-S-ylglycinyl)hydroquinone, (O, dashed line) 2-(cystein-S-yl)hydroquinone, (9, dashed line) 2-(N-acetylcystein-S-yl)hydroquinone. Each point represents the mean ( SEM (n ) 3). treated with either benzene (11.2 mmol/kg, sc in corn oil), 2-(GSyl)HQ (200 µmol/kg, iv), 2,6-bis(GSyl)HQ (50 µmol/kg, iv), or 2,3,5-tris(GSyl)HQ (17 mmol/kg, iv) on day 1 at 1 a.m. and 1 p.m. and on day 2 at 1 a.m. The rats were given 12 µCi of 59FeCl (ip) at 1 p.m. on day 3, and 24 h later blood was drawn 3 via cardiac puncture on anesthetized (equithesin) animals. Whole blood (200 µL; anticoagulant, EDTA) was washed twice with normal saline to remove plasma and analyzed using a gamma counter (1282 CompuGamma, LKB Wallac). Calculations for the amount of 59Fe uptake assumed a blood volume equivalent to 6% of body weight. Rats exhibiting signs of malabsorption of 59FeCl3 (counts per min ∼ background) were discarded from the analysis. Statistical Analyses. All data are expressed as mean ( standard error. Statistical analyses were performed using ANOVA with Scheffe’s post hoc analysis.

Results Identification of Quinol Thioethers in Bone Marrow of Rats and Mice Treated with Hydroquinone/ Phenol or Benzene. Following coadministration of hydroquinone/phenol (2.0 mmol/kg, ip) to rats, 2-(GSyl)HQ, 2,3,5-tris(GSyl)HQ, 2,5-bis(GSyl)HQ, and 2,6-bis(GSyl)HQ were all detected in bone marrow, reaching maximum values of 15.34 ( 1.3, 0.82 ( 0.08, 0.15 ( 0.03, and 0.58 ( 0.08 nmol/2 femurs, respectively, at 15 min (Figure 1A). Between 15 and 60 min, 2-(GSyl)HQ levels

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Figure 2. Identification and quantitation of quinol thioether metabolites in mouse bone marrow. Following coadministration of hydroquinone (0.9 mmol/kg, ip) and phenol (1.1 mmol/kg, ip), hydroquinone-glutathione conjugates (A) and mercapturic acid pathway metabolites (B) were quantified in mouse bone marrow by HPLC-CEAS: (A) (O) hydroquinone, (b) 2-(glutathion-S-yl)hydroquinone; (B) (b) 2-(glutathion-S-yl)hydroquinone, (0, dashed line) 2-(cystein-S-ylglycinyl)hydroquinone, (O, dashed line) 2-(cystein-S-yl)hydroquinone, (9, dashed line) 2-(N-acetylcystein-Syl)hydroquinone. Each point represents the mean ( SEM (n ) 3).

declined with the concomitant formation of the γ-GTcatalyzed metabolite, 2-(Cys-Gly)HQ, the dipeptidase metabolite, 2-(Cys)HQ, and the mercapturic acid metabolite, 2-(NAC)HQ. The latter three metabolites reached maximum levels of 0.15 ( 0.03, 3.49 ( 0.30, and 1.91 ( 0.79 nmol/2 femurs by 30 min, respectively (Figure 1B). 2-(Cys)HQ and 2-(NAC)HQ appeared to persist in bone marrow throughout the remainder of the experiment. A similar metabolic profile was observed in mice, with the exception that 2,5-bis(GSyl)HQ, 2,6-bis(GSyl)HQ, and 2,3,5-tris(GSyl)HQ were frequently not detectable (Figure 2). The latter conjugates were only observed in those mice exhibiting high concentrations of 2-(GSyl)HQ in marrow (data not shown). Following twice daily administration of benzene (11.2 mmol/kg) for 2 days, 2-(GSyl)HQ, 2-(Cys-Gly)HQ, 2-(Cys)HQ, and 2-(NAC)HQ were all detected in the bone marrow of rats and mice. The total amount of quinol thioethers detected was 4.1 ( 1.8 and 9.9 ( 4.1 pmol/mg of protein (mean ( SEM, n ) 3 and 5) in rats and mice, respectively. The presence of a functional mercapturic acid pathway in bone marrow of both species was confirmed by in vitro studies. Microsomal N-acetylation of 2-(Cys)HQ and cytosolic N-deacetylation of 2-(NAC)HQ were determined. The ratio of N-acetylation to N-deacetylation was 8.3 ( 1.2 and 21.0 ( 5.0 for rats and mice, respectively. Mice also exhibited the highest activities for both the cysteine conjugate N-acetyltransferase and the N-acetylcysteine conjugate N-deacetylase (Figure 3). Although the absolute amount of quinol thioethers present in bone marrow was highest in rats (Figures 1 and 2), the mice exhibited a 1.8-2.8-fold larger area under the bone marrow

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Figure 3. Cysteine conjugate N-acetyltransferase and Nacetylcysteine conjugate deacetylase activities in rat and mouse bone marrow. Microsomal N-acetyltransferase activity was determined with 2-(cystein-S-yl)hydroquinone as substrate, and cytosolic N-deacetylase activity was determined with 2-(Nacetylcystein-S-yl)hydroquinone as the substrate. Values are expressed as fmol (µg of protein)-1 min-1 and represent the mean ( SEM (n ) 5).

Bratton et al.

Figure 5. Uptake of intravenously administered 2-(glutathionS-yl)hydroquinone into rat bone marrow. Following administration of 2-(glutathion-S-yl)hydroquinone (100 µmol/kg, iv) to rats, the quantities of (b) 2-(glutathion-S-yl)hydroquinone and its metabolites, (9, dashed line) 2-(N-acetylcystein-S-yl)hydroquinone, (O, dashed line) 2-(cystein-S-yl)hydroquinone, and (0, dashed line) 2-(cystein-S-ylglycinyl)hydroquinone, in bone marrow were determined by HPLC-CEAS. Each point represents the mean ( SEM (n ) 3).

Table 1. Estimated Concentrations of Quinol Thioethers in Bone Marrow following Coadministration of Hydroquinone/Phenol time at Cmax (min) quinol thioether Cmax (nmol) femur volume (µL) (×2) concentration (µM)

Figure 4. Formation and elimination of quinol thioether metabolites in rat and mouse bone marrow. Quinol thioether formation and elimination were compared in (O) rats and (b) mice, following coadministration of hydroquinone (0.9 mmol/ kg, ip) and phenol (1.1 mmol/kg, ip). Data points represent the sum total of quinol thioethers (pmol/µg of protein) present in the bone marrow and are expressed as the mean ( SEM (n ) 3).

concentration-time curve (AUC) when the data were expressed on a pmol/µg of protein basis. Using this normalized data, quinol thioether peak concentrations were higher and more slowly eliminated in mice than in rats (Figure 4). Peak quinol thioether concentrations were also calculated on a nmol/vol basis using estimated femur volumes of 50-75 and 4-5 µL for rats and mice, respectively. For both species, bone marrow concentrations of quinol thioethers were in excess of 180 µM (Table 1). Although lower levels of the quinol thioethers were detected following benzene administration, concentrations in mouse marrow were higher than those in the rat (see above). Moreover, it is unlikely that our estimates reflect the Cmax for quinol thioethers in bone marrow following benzene adminstration, because the optimal dosing regimen to achieve maximal quinol thioether concentrations has not been determined, nor has the time point at which Cmax occurs been identified. Limited Uptake of 2-(GSyl)HQ from the Circulation into Bone Marrow. Following intravenous ad-

SD rats

DBA/2 mice

15 19.4 ( 1.8 ∼100 ∼194

30 1.5 ( 0.2 ∼8 ∼188

ministration of 2-(GSyl)HQ (100 µmol/kg) to male Sprague-Dawley rats, 2-(GSyl)HQ, 2-(Cys-Gly)HQ, 2-(Cys)HQ, and 2-(NAC)HQ were all identified in bone marrow (Figure 5). The maximum concentration (0.56 ( 0.37 pmol/µg of protein) of quinol thioethers was reached at 2.5 min, and the AUC was between 12 and 14 pmol‚min/ µg of protein. However, administration of 2-(GSyl)HQ rarely resulted in the presence of multisubstituted GSH conjugates in bone marrow, and the total amount of quinol thioethers isolated (4.6 ( 3.1 nmol/2 femurs) at the peak time point represented only 0.023 ( 0.016% of the administered dose. In contrast, following coadministration of hydroquinone/phenol (2.0 mmol/kg, ip), the maximum concentration of quinol thioethers (3.1 ( 0.4 pmol/µg of protein) was reached at 15 min and included 2-(GSyl)HQ, 2-(Cys-Gly)HQ, 2-(Cys)HQ, 2-(NAC)HQ, 2,5bis(GSyl)HQ, 2,6-bis(GSyl)HQ, and 2,3,5-tris(GSyl)HQ. In addition, the AUC was between 121 and 160 pmol‚ min/µg of protein (Figure 6). Hematotoxicity of Hydroquinone-Glutathione Conjugates. Administration of either 2,3,5-tris(GSyl)HQ (17 µmol/kg, iv), 2,6-bis(GSyl)HQ (50 µmol/kg, iv), or benzene (11.2 mmol/kg, sc in corn oil) to rats significantly reduced 59Fe incorporation into immature erythrocytes (pronormoblasts and normoblasts) to 44.5 ( 6.0%, 28.3 ( 3.4%, and 20.3 ( 8.5% of control values, respectively (p < 0.05) (Figure 7). Although the doses of 2,3,5tris(GSyl)HQ and 2,6-bis(GSyl)HQ represent only 0.2% and 0.4% of the dose of benzene, there was no statistical difference between these compounds in their ability to damage bone marrow (pro)normoblasts and subsequently reduce the reticulocyte pool available for 59Fe incorporation. In contrast, 2-(GSyl)HQ (200 µmol/kg, iv) had little effect on 59Fe uptake at the dose tested.

Quinol Thioether-Mediated Hematotoxicity

Figure 6. Pharmacokinetics of quinol thioethers in rat bone marrow following administration of hydroquinone (0.9 mmol/ kg, ip) and phenol (1.1 mmol/kg, ip) or 2-(glutathion-S-yl)hydroquinone (100 µmol/kg, iv). Following administration of (O) hydroquinone (0.9 mmol/kg, ip) and phenol (1.1 mmol/kg, ip) or (b) 2-(glutathion-S-yl)hydroquinone (100 µmol/kg, iv), the total amount of quinol thioethers present in bone marrow was determined by HPLC-CEAS. Each point represents the mean ( (SEM (n ) 3).

Figure 7. Effect of benzene, 2-(glutathion-S-yl)hydroquinone, 2,6-bis(glutathion-S-yl)hydroquinone, and 2,3,5-tris(glutathionS-yl)hydroquinone on 59Fe uptake into rat hemoglobin. Male Sprague-Dawley rats were given three doses of either 2-(glutathion-S-yl)hydroquinone (200 µmol/kg, iv, open bar), 2,6-bis(glutathion-S-yl)hydroquinone (50 µmol/kg, iv, light-hatched bar), 2,3,5-tris(glutathion-S-yl)hydroquinone (17 µmol/kg, iv, horizontally-lined bar), or benzene (11.2 mmol/kg, sc in corn oil, dark-hatched bar) within the first 24 h of the experiment. Control animals received PBS (black bar). 59FeCl3 (12 µCi/rat, ip) was then administered 48 h later, and after a further 24 h, blood samples were obtained and processed as described in Materials and Methods. Each bar represents the mean ( SEM (n ) 5). Statistical significance was determined by one-way ANOVA with Scheffe’s post hoc analysis. *Significantly different from control values at p < 0.05.

Discussion Thioether metabolites of hydroquinone were identified in the bone marrow of male Sprague-Dawley rats and DBA/2 mice following either hydroquinone/phenol (2.0 mmol/kg, ip) (Figures 1 and 2) or benzene (4 × 11.2 mmol/ kg, sc) administration. Concentrations (pmol/µg of protein) of quinol thioethers in bone marrow were higher in mice than in rats of hydroquinone/phenol-treated animals (Figure 4), which correlates with the relative sensitivity of these two species to benzene-induced hematotoxicity (7). Similar results were obtained in benzene-treated animals. Both rats and mice produce significant amounts of 2-(GSyl)HQ (Figures 1A and 2A), following hydroquinone/phenol administration, but 2,5-bis(GSyl)HQ, 2,6bis(GSyl)HQ, and 2,3,5-tris(GSyl)HQ were only detected in the bone marrow of rats (Figures 1A and 2A). In contrast, the latter conjugates could not be consistently identified in mouse bone marrow, and several reasons may account for this finding. First, we estimate rat and mouse femur volumes to be approximately 50-75 and 4-5 µL, respectively; however, the minimal volumes used

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to flush bone marrow from each species were 1.0 and 0.5 mL, respectively. Thus, bone marrow isolated from mice is 5-fold more dilute than bone marrow isolated from rats. In addition, bone marrow cells from mice may be more susceptible to arylation by quinol thioethers since bone marrow stromal cells isolated from mice exhibit 28-fold lower DT-diaphorase (NAD(P)H quinone oxidoreductase) activity than those isolated from rats (33). Bone marrow cells exhibiting high peroxidase activity relative to DTdiaphorase activity yield more 1,4-benzoquinone-derived protein adducts (34) and by extension may represent cellular targets for the thioether metabolites of hydroquinone. Thus, a greater fraction of the multisubstituted GSH conjugates in mouse bone marrow may be adducted to proteins, limiting the "free" fraction available for detection by HPLC. Indeed, GSH conjugates of hydroquinone are more chemically (re)active than hydroquinone and are capable of arylating tissue macromolecules (26, 28, 29). For example, the kidney is a target of quinone-thioether toxicity, and nearly all of a dose of 2,3,5-tris(GSyl)[14C]HQ perfused into the renal artery of rats becomes covalently bound to renal proteins (29). Therefore, in both rats and mice, the amount of multisubstituted GSH conjugates of hydroquinone detected in bone marrow likely represents only a fraction of the total produced. The γ-GT- and dipeptidase-catalyzed products of 2-(GSyl)HQ, 2-(Cys-Gly)HQ and 2-(Cys)HQ, were also identified in the bone marrow of rats and mice. Concentrations of the cysteine conjugate are relatively high and persist in bone marrow (Figures 1B and 2B). The availability of bone marrow γ-GT and dipeptidases for processing of GSH conjugates is important, because cysteinyl-glycine and cysteine conjugates of hydroquinone are more chemically (re)active than their corresponding GSH conjugates (29). Consequently, such metabolites will be more potent arylators and redox cyclers. The half-wave oxidation potentials of the GSH conjugates of hydroquinone are also considerably lower than the half-wave oxidation potential of hydroquinone (28, 29). In addition, the conjugates are also more readily oxidized by cytochrome(s) P450 to the corresponding quinones than hydroquinone2 and generate more superoxide anions.3 Interestingly, 2-bromo[14C]hydroquinone (0.8 mmol/kg, ip) gives rise to radiolabeled protein adducts in both bone marrow and spleen, a fraction of which is inhibited by pretreating animals with acivicin (10 mg/kg) to inhibit γ-GT. Quinone thioethers therefore probably contribute to the covalent binding of 2-bromo[14C]hydroquinone in these tissues (35). The mercapturic acid 2-(NAC)HQ was also detected in bone marrow, suggesting the presence of a functional mercapturic acid pathway in both species. This was confirmed by demonstrating the ability of rat bone marrow microsomes and cytosol to catalyze the Nacetylation of 2-(Cys)HQ and N-deacetylation of 2-(NAC)HQ, respectively (Figure 3). N-Acetylation predominated in both species, but mice exhibited greater N-acetyltranferase and N-deacetylase activity than rats. Because the microsomal and cytosolic fractions used in these experiments were isolated from whole bone marrow homogenates, it is possible that specific cell types within bone marrow exhibit differences in their ability to N-acetylate 2 3

Sawalha, A., Monks, T. J., and Lau, S. S. Unpublished data. Butterworth, M., Lau, S. S., and Monks, T. J. Unpublished data.

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reactive cysteine conjugates, and such cells may represent susceptible targets for these metabolites. The liver produces significant amounts of the multisubstituted GSH conjugates following administration of hydroquinone (1.8 mmol/kg) (28); therefore, it seems plausible that the conjugates identified in the bone marrow might be transported there via the circulation. However, an intravenous dose of 2-(GSyl)HQ (100 µmol/ kg) yields quinol thioether concentrations in bone marrow which are far below those detected following coadministration of hydroquinone/phenol (2.0 mmol/kg, ip) (Figure 6). The ∼10-fold difference in AUCs suggests that the major fraction of the GSH conjugates present in bone marrow is formed in situ. Myeloperoxidase- and prostaglandin H synthase-mediated oxidation of hydroquinone to 1,4-benzoquinone, in the presence of GSH, has been shown to yield 2-(GSyl)HQ in vitro (35); therefore, this reaction should occur in bone marrow which contains both enzymes (36, 37). If concentrations of 2-(GSyl)HQ saturate bone marrow γ-GT (km ) 68 µM), subsequent oxidation and GSH substitution will lead to the formation of multisubstituted GSH conjugates of hydroquinone, including 2,6-bis(GSyl)HQ and 2,3,5-tris(GSyl)HQ. Since our estimate of quinol thioether concentrations in bone marrow exceeds 180 µM (Table 1), this metabolic pathway should occur readily in vivo. This view is supported by the identification of 2,5-bis(GSyl)HQ, 2,6-bis(GSyl)HQ, and 2,3,5-tris(GSyl)HQ in the bone marrow of rats following coadministration of hydroquinone/phenol. 2,3,5-Tris(GSyl)HQ and 2,6-bis(GSyl)HQ are efficient generators of reactive oxygen species.3 They are also toxic to (pro)normoblasts (59Fe incorporation assay) at doses of 17 and 50 µmol/kg (iv), respectively (Figure 7), which represent only 0.2% and 0.4% of the dose of benzene required to produce a similar level of toxicity. In addition, because