Oxidation and Acetylation as Determinants of 2-Bromocystein-S-ylhydroquinone-Mediated Nephrotoxicity

Chem. Res. Toxicol. 1994, 7, 495-502 496

Oxidation and Acetylation as Determinants of 2-Bromocystein-S-ylhydroquinone-Mediated Nephrotoxicity

Terrence J. Monks,* Herng-Hsiang Lo,+ and Serrine S. Lau Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin,

Austin, Texas 78712

Received February 8, 1994'

2-Bromodiglutathion-S-ylhydroquinone is a more potent nephrotoxicant than 2-bromomonoglutathion-S-ylhydroquinones. In the present study we examined the activity of enzymes involved in mercapturic acid biosynthesis toward both the glutathione conjugates and their cysteine and N-acetylcysteine metabolites and compared the results to the relative nephrotoxicity of these conjugates. Although differences were observed in the kinetics of the y-glutamyl transpeptidase (y-GT)-mediated hydrolysis and transpeptidation of the glutathione conjugates, the concentration of this enzyme within the kidney probably precludes it from contributing to their differential toxicity. In contrast, the rate a t which the cysteine and corresponding mercapturate conjugates underwent N - deacetylationlN-acetylation cycling correlated with previously reported differences in toxicity. The relative rates of these two reactions are important because electrochemical data suggest that 2-bromodicystein-S-ylhydroquinone is more readily oxidized to the reactive quinone than its corresponding mercapturic acid. In addition, 2-bromodi(N-acetylcystein-S-yl)hydroquinone, which is the most potent of the mercapturic acid conjugates, exhibited the highest N-deacetylationlN-acetylation ratio. In contrast, 2-bromo-3-(N-acetylcystein-S-y1)hydroquinone, which is essentially not toxic in vivo, was not a substrate for the renal cysteine conjugate N-deacetylase. The data suggest that the rate-determining step for the in vivo toxicity of these conjugates is probably the N-acetylation reaction and the availability of the corresponding acetyl-coA cofactor.

Introduction The conjugation of glutathione (GSH)' with a variety

of chemicals, and/or their reactive metabolites, usually results in the detoxication of reactive electrophiles and facilitates their excretion into urine as their corresponding mercapturic acids. Recently, however, several examples have been reported where conjugation of quinones with GSH fails to eliminate their biological or toxicological reactivity (reviewed in ref 1) probably in part because quinone thioethers maintain the ability to redox cycle with the concomitant generation of reactive oxygen species (2, 3). Quinone thioethers have also been shown to be substrates for and inhibitors of a variety of enzymes that utilize either quinones and/or GSH as substrates and cosubstrates, respectively (4-1 1). The kidney appears to be particularly susceptible to the toxicity of quinone thioethers. For example, the nephrotoxicity of bromobenzene is probably due to the formation of 2-bromodiglutathion-S-ylhydroquinone [BBr-(diGSyl)HQ] and three positional isomers of 2-bromomonoglutathion-S-ylhydroquinone (12). Thus, whereas bromobenzene causes

* To whom correspondence should be addressed. Tel: (512) 471-6699;

t Present address: University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, TX 78957.

* Abstract publiihed in Advance ACS Abstracts, May 15, 1994. 1 Abbreviations: glutathione [GSH]; 2-bromodiglutathion-S-ylhy-

droquinone [2-Br-(diGSyl)HQ]; 2-bromomonoglutathion-S-ylhydroquinones [P-Br-&(GSyl)HQ, 2-Br-5-(GSyl)HQ, 2-Br-6-(GSyl)HQ] ; 2-bromodi(N-acetylcystein-S-y1)hydroquinone [2-Br-(diNAC)HQI ; 2-bromo- 3-(N-acetylcystein-S-yl)hydroquinone [2-Br-3-(NAC)HQI; 2-bromo-BfN- acetylcystein-S-y1)hydroquinone [2-Br-5-(NAC)HQI; 2-bromo-6-(N- acetylcystein-S-y1)hydroquinone [2-Br-6-(NAC)HQI; 2-bromodicystein- S-ylhydroquinone [2-Br-(diCYS)HB]; 2-bromo-Scystein-S-ylhydrcq~one [2-Br-3-(CYS)HQ]; 2-bromo-5-cystein-S-ylhydrcquinone [2-Br-S-(CYS)- HQ]; 2-bromo-6-cystein-S-ylhydroquinone [2-Br-6-(CYS)HQI; 2-bromohydroquinone [2-BrHQ]; y-glutamyl transpeptidase [y-GT].

FAX: (512) 471-6002.

0893-228~/94/2707-0495$04.50JQ

nephrotoxicity in the rat at a dose of 9.3 mmol/kg (13), 2-Br-(diGSyl)HQ causes enzymuria, proteinuria, and glucosuria at a dose of only 10 pmol/kg (14). The nephrotoxicity of 4-aminophenol in rats may similarly be due to metabolism to the quinone imine followed by GSH conjugation. In support of this view, Fowler et al. (15) have shown that 4-amino-3-glutathion-S-ylphenol repro- duces 4-aminophenol nephrotoxicity, but at doses 3-4- fold lower. In addition, 4-amin0-2-glutathion-S-ylpheno1, 4amino-3-glutathion-S-ylphenol,4-amino-2,5diglutathion- S-ylphenol, and 4-amino-2,3,5(or 6)-triglutathion-S- ylphenol have been identified in the bile of Wistar rata following administration of 0.92 mmol/kg 4-aminophenol (1 6), and the latter three conjugates all caused cytotoxicity when incubated with freshly isolated rat kidney cortical cells (16). Furthermore, 2,5-dichloro-3-glutathion-S-yl- 1,Cbenzoquinone (1 3, 2,5,6-trichloro-3-glutathion-S-yl- 1,Cbenzoquinone (1 3, 2,3-diglutathion-S-y1-1,4-naphthoquinone (18), 2-glutathion-S-yl-l,4-naphthoquinone (18), and the mercapturic acid of menadione (but not the GSH conjugate) (18) are also moderately nephrotoxic following administration (200 pmol/kg) to rats.

In contrast to the acute nephrotoxic effects of quinone thioethers, they may also play an important role in the nephrocarcinogenicity of several phenolic compounds (1 ). For example, long-term exposure to hydroquinone causes marked increases in renal tubular cell adenomas in Fischer 344 rats (19,20), and 2,3,5-triglutathion-S-ylhydroquinone, 2,5-diglutathion-S-ylhydroquinone, 2,6-diglutathion-S-y1- hydroquinone, and 2-glutathion-S-ylhydroquinone, which are all in vivo metabolites of hydroquinone (21), cause renal proximal tubular cell necrosis at comparatively low doses (10-50 pmol/ kg) (22). The ability of quinone thioethers to react with DNA either directly (23) or

0 1994 American Chemical Society

496 Chem. Res. Toxicol., Vol. 7, No. 4, 1994 Monks et al.

absorbing peak and were used for NMR structural analysis, and revealed that product 1 was 2-bromodicystein-S-ylhydroquinone [a-Br-(diCYS)HQ], product 2 was 2-bromo-3-cystein-S-ylhydroquinone [2-Br-3-(CYS)HQI, and product 3 was an ap- proximately 1:2 mixture of 2-bromo-5-cystein-S-ylhydroquinone [2-Br-5-(CYS)HQl and 2-bromo-6-cystein-S-ylhydroquinone [2-Br-6-(CYS)HQ] by the following criteria. The presence of the cysteine residue in each compound was shown by the characteristic groupings at high field. The positions of the hydroquinone hydroxyl groups are already established; i.e., carbons 1 and 4. 2-Bromo-3-cystein-S-ylhydroquinone [2-Br- 3-(CYS)HQ]: ‘H, 6.97 and 6.89 ppm (H5 and H6; J = 8.9 Hz, i.e., ortho coupling); 3.58 (lH, dd, Cys-a); 3.40 and 3.13 (2H, dd’s, Cys-8). 2-Bromo-5-cystein-S-ylhydroquinone and 2-bromo- 5-cystein-Sylhydroquinone: lH, 7.12 and 7.08 ppm (2H, s; J I 1 Hz, Le., para-coupled (H3 and H6) protons); 7.05 and 6.98 (2H, d; J = 3.0 Hz; Le., meta-coupled (H3 and H5) protons). The position of cysteine substitution in these two compounds was therefore at carbons 5 and 6, respectively, with signals attributable to cysteine at 3.77 and 3.74 ppm (2H, dd’s, Cys-a’s) and 3.48, 3.46,3.21, and 3.17 ppm (4H, dd’s, Cys-ps). The intensity of the signals associated with the two compounds suggested an ap- proximately 1:2 ratio of 2-Br-5-(CYS)HQ and 2-Br-6-(CYS)HQ. 2-Bromodicystein-Sylhydroquinone [2-Br-(diCYS)HQl: ‘H, 7.09 ppm (single aromatic, indicating the disubstituted nature of this compound). Moreover, the signals corresponding to the cysteine protons [3.89 and 3.75 ppm (2H, dd’s, Cys-a’s); 3.50 and 3.37 (2H, dd’s, Cys-ps); 3.31 (2H, m, Cys-ps, obscured by CD3- OH)] exhibited a more complex pattern, and integration of the signals corresponding to the characteristic high-field aliphatic cysteine protons also confirmed the addition of two molecules of cysteine.

Synthesis and Purification of 2-Bromo(N-acetylcystein- S-y1)hydroquinones. 2-Bromo-1,4-quinone (1.25 M 2.66 mL of methanol) was added dropwise to a solution of N-acetyl+ cysteine (50 mM; 132 mL of distilled water) and the mixture stirred at room temperature for 2 h. The reaction mixture was then frozen in dry ice/acetone and lyophilized overnight. The resulting product was purified by HPLC by dissolving in distilled water (100 mg/mL) and injecting 200-pL aliquots onto a 5-pm Ultrasphere ODS (Beckman) reverse-phase semipreparative column (25 cm X 10 mm). The sample was eluted with an isocratic mixture of water/methanol/acetic acid (7623:l v/v) at a flow rate of 3 mL/min for 40 min and the eluate monitored at 280 nm. Four major UV-absorbing products were eluted from the column with retention times of 20.1 (product l ) , 23.9 (product 2), 26.8 (product 3), and 37.7 min (product 4). Each product was collected from several injections of the lyophilized synthetic material. The methanol component of each peak was removed by rotary evaporation under vacuum, and the remaining aqueous fractions were frozen in dry ice/acetone and lyophilized. Each of the resulting powders, when reanalyzed by analytical HPLC, gave rise to a single UV-absorbing peak. This material was therefore used for structural analysis by NMR spectroscopy, which revealed that product 1 was 2-bromo-3-(N-acetylcystein-S-y1)hydroquinone [2-Br-3-(NAC)HQl, product 2 was 2-bromo-B-(N- acetylcystein-S-y1)hydroquinone [2-Br-5-(NAC)HQ], product 3 was 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone [2-Br-6- (NAC)HQ] , and product 4 was 2-bromodi(N-acetylcystein-S- y1)hydroquinone [2-Br-(diNAC)HQ] by the following criteria. The presence of the N-acetylcysteine residue in each compound was shown by the characteristic groupings at high field. The positions of the hydroquinone hydroxyl groups are already established; i.e., carbons 1 and 4. 2-Bromo-3-(N-acetylcystein- S-y1)hydroquinone [2-Br-3-(NAC)HQJ: ‘H, 6.72 and6.63 ppm (H5 and H6; J = 8.86 Hz, Le., ortho coupling); 4.13 (lH, dd, Cys-a); 3.26and 3.02 (2H, dd‘s, Cys-8); 1.67 (3H,s, CH3-N-acetyl). 2-Bromo-5-(N-acetylcystein-S-yl)hydroquinone [2-Br-5- (NAC)HQI: ‘H, 6.95 and 6.86 ppm (H3 and H6; J > 1 Hz, Le., para coupling); 4.33 (lH, dd, Cys-a); 3.25 and 3.07 (2H, dd’s, Cys-8); 1.74 (3H, 8 , CHs-N-acetyl). 2-Bromo-6-(N-acetylcystein-S-y1)hydroquinone [2-Brb6-(NAC)HQ]: ‘H, 6.84 and 6.71

indirectly via reactive oxygen generation (24) may contribute to the carcinogenic activity of these compounds. 2-Bromomonoglutathion-S-ylhydroquinones are sub-

stantially less toxic than 2-Br-(diGSyl)HQ, and differences in the position of GSH addition also affect the relative toxicity of the 2-Br-(monoGSyl)HQ’s (25). 2,3,5-Triglu- tathion-S-ylhydroquinone is also a more potent nephrotoxicant than either 2,3-diglutathion-S-ylhydroquinone, 2,5-diglutathion-S-ylhydroquinone, 2,6-diglutathion-S-y1- hydroquinone, or 2-glutathion-S-ylhydroquinone (22), and 2,3-diglutathion-S-yl-l,4-naphthoquinone is more toxic than 2-glutathion-S-yl-l,4-naphthoquinone (181, although the reasons for such differences in toxicity are unclear. Inhibition of renal y-glutamyl transpeptidase (yGT) by pretreatment of animals with acivicin significantly reduced the nephrotoxicity caused by both a-Br-(diGSyl)HQ and 2,3,5-triglutathion-S-ylhydroquinone, indicating that metabolism by y-GT is required for toxicity (14, 22). Therefore, differences in the metabolism of these conjugates by y-GT, and/or in the metabolism of the corresponding cystein-S-yl and N-acetylcystein-S-yl conjugates, might be important determinants of toxicity. We have therefore investigated the metabolism of 2-Br- (diGSyl)HQ, 2-Br-(monoGSyl)HQ’s, and the corresponding cystein-S-yl and N-acetylcystein-S-yl conjugates by the enzymes of the mercapturic acid pathway. The results suggest that an important determinant of toxicity is the relative rate at which the cystein-S-yl and N-acetylcystein- S-yl conjugates undergo N-acetylationlN-deacetylation cycling.

Materials and Methods

Caution: 2-Bromohydroquinone (2-BrHQ) is nephrotoxic in rats and must be handled with care. 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.

Chemicals. 2-BrHQ was a product of ICN (Cleveland, OH), and 2-bromo-1,4-quinone was prepared according to previously published procedures (12). Cysteine, N-acetylcysteine, ascorbic acid, and acetyl coenzyme A were purchased from the Sigma Chemical Co. (St. Louis, MO). All other reagents were of the highest grade commercially available.

Synthesis and Purification of 2-Bromocystein-5-ylhydroquinones. 2-Bromo-l,4-quinone (1.16 M in 2.5 mL of methanol) was added dropwise to a solution of 132 mL of L-cysteine (50 mM) in distilled water and the mixture stirred for 30 min at room temperature. The reaction mixture was filtered (Acro LC 25,0.45 pm), frozen in dry ice/acetone, and lyophilized overnight to give a white powdery product. Portions (20 mg) of the product were then dissolved in MezS0/0.9 % saline/acetic acid (400 pL/400 pL/8 pL) and purified by HPLC (Shimadzu Model LC6A chromatograph equipped with a UV/vis variable- wavelength spectrophotometric detector, Model SPD-6AV). The sample was injected onto a system containing two Partisil Magnum 9 ODs-3 (10 pm, 25 cm X 9.4 mm) reverse-phase semipreparative columns (Whatman) connected in series and eluted with an isocratic mixture of water/methanol/acetic acid (79201 v/v over 30 min) at a flow rate of 3 mL/min, and the eluate was monitored at 280 nm. Under these conditions, three major UV-absorbing products were eluted from the column with retention times of 19.1 (product I) , 21.6 (product 2), and 27.1 min (product 3). Each product was collected from several injections of the lyophilized synthetic material. The methanol was then removed by rotary evaporation under vacuum (Buchi RE-121A), and the remaining aqueous fractions were frozen in dry ice/ethanol and lyophilized. The resulting white powders, when reanalyzed by HPLC, each gave rise to a single UV-

Determinants of Quinone Thioether-Mediated Toxicity

ppm (C3 and C5; J = 2.9 Hz, i.e., meta coupling); 4.26 (lH, dd, Cys-a); 3.20and 3.00 (2H, dd's, Cys-(3); 1.68 (3H, s, CHa-N-acetyl). 2-Bromodi(N-acetylcystein-S-yl)hydroquinone [2-Br-(di- NAC)HQ]: 'H, 6.80 ppm (single aromatic, indicating the disubstituted nature of this compound). This was substantiated by the presence of resonances consistent with the presence of twoN-acetylcysteine residues: 4.27 and 4.125 ppm (2H, dd, Cys- a's); 3.26, 3.23, 3.05, and 3.02 (4H, dt's, Cys-j3's); 1.68 and 1.67 (6H, s, CHB-N-acetyl).

Preparation of Cytosolic and Microsomal Fractions. Animals were euthanized by cervical dislocation. The livers and the kidneys were removed, and the cortex was dissected from the rest of the kidney. The tissues were homogenized with Tris-KC1 buffer (pH 7.4; 0.15 M KCl; 20 mM *is) at 4 OC (1:5 w/v). The homogenates were centrifuged at loooOg for 20 min. The loooOg supernatants were further centriguged at lOOOOOg for 1 h toobtain the microsomal and cytosolic fractions.

Biosynthesis of 2-Bromo-5-cystein-Sy1 hydroquinone and 2-Bromo-6-cystein-Sylhydroquinone. Preparative chromatographic resolution of 2-Br-5-(CYS)HQ and 2-Br-&(CYS)HQcould not be achieved under conditions employed in the present studies. However, to enable quantitation of the in vitro rates of N- deacetylation and N-acetylation (see below) and for the electrochemical studies, pure samples of these isomers were bio- synthesized from the corresponding authentic N-acetylcysteine analogs. 2-Br-5-(NAC)HQ or 2-Br-64NAC)HQ (0.62 mM) were incubated with 5 mg/mL rat kidney l00OOOg supernatant in 0.1 M phosphate buffer (pH 7.4) in the presence of 2.2 mM ascorbic acid (to prevent oxidation and cyclization of the product) in a final volume of 46 mL, for 60 min at 37 OC. The reaction was terminated by the addition of 4.6 mL of 10% perchloric acid, vortex-mixed, and placed on ice for 10 min. The mixture was centrifuged to remove precipitated protein, and the supernatant was frozen in dry ice/acetone. Lyophilization gave rise to a milky white suspension (-8 mL) that was centrifuged to yield a clear yellow supernatant, to which was added 10 N NaOH to a pH of 1.9. Aliquots of the supernatant were then purified by semipreparative HPLC. The mobile phase consisted of methanol/ water/acetic acid (5:941 v/v) for 5 min followed by a 25-min linear gradient to 100% methanol, at a flow rate of 3 mL/min. The eluate was monitored at 310 nm. Under these conditions 2-Br-5-(CYS)HQ and 2-Br-5-(NAC)HQ, and 2-Br-6-(CYS)HQ and 2-Br-6-(NAC)HQ, eluted with retention times of 17.8 and 22.7 min, and 17.6 and 23.0 min, respectively. The peaks corresponding to 2-Br-5-(CYS)HQ and 2-Br-6-(CYS)HQ were collected from successive HPLC runs, the methanol content was removed by rotary evaporation under vacuum, and the residual aqueous fraction was frozen in dry ice/acetone and lyophilized.

Kinetics of the y-Glutamyl Transpeptidase-Mediated Hydrolysis and Transpeptidation of 2-Bromoglutathion- S-ylhydroquinones. 2-Bromodiglutathion-S-ylhydroquinone, 2-bromo-3-glutathion-S-ylhydroquinone, 2-bromo-5-glutathion- S-ylhydroquinone, and 2-bromc-6-glutathion-S-ylhydroquinone were prepared as previously described (12,26). Each substrate (2-160 pM) was incubated in 0.1 M Tris buffer (pH 7.4) containing 0.5 mM ascorbate, at 37 OC for 30 min, in the presence of 1.6 units/pL 12-Br-(monoGSy1)HQI or 8 units/pL [P-Br-(diGSyl)- HQ] rat renal y- GT. Incubations were terminated by the addition of 100 pL of 4.5% acetic acid, immediate freezing in a dry ice/ acetone bath and storage at -20 "C until analysis. For the transpeptidation reaction, glycylglycine (2 mM) was used as the y-glutamate acceptor and the incubation time shortened to 5 min for the 2-bromomonoglutathion-S-ylhydroquinones to maintain linear reaction conditions.

N-Deacetylation of 2-Bromo(N-acetyl-~-cystein-S-y1)- hydroquinones. Incubation mixtures contained 5 mg/mL liver or kidney cytosolic or microsomal protein, 1 mM ascorbic acid, and substrate at concentrations between 10 and 100 pM, in 0.1 M phosphate buffer (pH 7.4). The mixtures were incubated at 37 OC for 10 min. Control incubations contained boiled tissue preparations. Incubations were terminated by the addition of 0.1 mL of 10% perchloric acid to 1-mL aliquots of the incubation

Chem. Res. Toxicol., Vol. 7, No. 4, 1994 497

mixture. 2-BrHQ (50 nmol in 9.5 pL of MeOH) was added as the internal standard. The mixtures were placed on ice for 10 min and then centrifuged to remove denatured protein. The acidic supernatants were stored frozen at -20 "C until analysis. Aliquots (100 pL) of samples were injected onto a Partisil5 ODs-3 reverse- phase column (25 cm x 4.6 mm; Whatman) and eluted with a mobile phase of methanol/water/acetic acid (5941 v/v) for 5 min, followed by a 25-min linear gradient to 100% methanol, at a flow rate of 1 mL/min. The eluate was monitored at 310 nm. Under these conditions, the retention times (in parentheses) of the following pairs of thioethers were as follows: 2-Br-(diCYS)- HQ and 2-Br-(diNAC)HQ (15.5 and 22.6 min); 2-Br-3-(CYS)HQ and 2-Br-3-(NAC)HQ (15.9 and 19.3 min); 2-Br-54CYS)HQ and 2-Br-5-(NAC)HQ (17.0 and 21.5 min); and 2-Br-6-(CYS)HQ and 2-Br-6-(NAC)HQ (17.0 and 21.9 min). The internal standard (2-BrHQ) eluted with a retention time of 18.4 min. For quantitation, the peak area ratios of the metabolites to the internal standard were determined. With the exception of a-Br-(diCYS)- HQ, standard curves were prepared for each metabolite in the range 12.5-150 nmol. Deacetylation of 2-Br-(diNAC)HQ was quantified by determining the disappearance of the substrate, 2-Br-(diNAC)HQ, since three possible products of this reaction are possible, namely, a-Br-(diCYS)HQ and the two possible isomers of the disubstituted mixed thioether, 2-Br-(monoCYS- monoNAC)HQ. Authentic standards of the mixed thioethers are not available at present.

Renal Microsomal N-Acetylation of 2-Bromocystein-S- ylhydroquinones. The N-acetylation of 2-Br-(CYS)HQ conjugates was determined in freshly prepared rat renal microsomes with 100 pM substrate, 200 pM CoASAc, 1 mM ascorbic acid, and 1 mg of microsomal protein, in a final volume of 0.5 mL of 0.1 M potassium phosphate buffer (pH 7.0) at 37 OC. The mixtures were preincubated at 37 "C for 2 min and the reactions initiated by the addition of the CoASAc, and incubation was continued for a further 5 min. The reactions were terminated by the addition of 1.5 mL of 1.33 M acetic acid. The mixtures were vortex-mixed for 10 s, placed on ice for 10 min, and centrifuged to remove denatured protein. The acidic super- nantants were then analyzed by HPLC as described above.

Electrochemistry of 2-Bromohydroquinone Thioethers. Oxidation potentials were determined by HPLC (Shimadzu LC 6A) with coulometric (electrochemical) detection (ESA Cou- lochem Model 5100A). The detector was equipped with two porous graphite test electrodes connected in series. The applied potential at the second test electrode was varied from 4 . 2 to +0.6 V. Each of the 2-BrHQ-thioethers was prepared at a concentration of 5 mM in distilled water, and a 10-pL aliquot was injected onto a Partisil5 ODs-3 (25 cm X 4.6 mm; Whatman) reverse-phase column and eluted with 12.5 mM citrate/25 mM ammonium acetate (pH 4.0) containing 25% methanol and 20 mg/L EDTA, at a flow rate of 1 mL/min. Peak areas were measured at each potential and expressed as a percentage of the maximum response obtained. Hydrodynamic voltammograms for each of the 2-BrHQ-thioethers were prepared, and the half- wave oxidation potentials (&p) were determined.

Results

Kinetics of the y-Glutamyl Transpeptidase-Medi- ated Hydrolysis and Transpeptidation of 2-Bromo- glutathion-S-ylhydroquinones. Lineweavel-Burk ploh for t he 7-GT-mediated hydrolysis and transpeptidation of 2-Br-3-(GSyl)HQ, 2-Br-5-(GSyl)HQ, 2-Br-6-(GSyl)HQ, and 2-Br-(diGSyl)HQ revealed significant differences between the kinetics of t he metabolism of t he 2-Br- (monoGSy1)HQ conjugates and of 2-Br-(diGSyl)HQ (Fig- ure 1). The K m and Vm, for t he hydrolysis of 2-Br-3- (GSyl)HQ, 2-Br-5-(GSyl)HQ, and 2-Br-6-(GSyl)HQ were identical [26.4 f 2 p M and 13.7 f 0.6 pmol/(unit.min), respectively] whereas the Km values for the transpeptidation reaction (with glycylglycine as the acceptor) were

498 Chem. Res. Toxicol., Vol. 7, No. 4, 1994 Monks et al.

1N

15001

/ Transpeptidase ;o Hyplase I /y 2-Br-(diGSyl)HQ

/ f 5w 2-Br-mono-(GSyl)HQ’s

f -cf8 Transpeptidase

n -0.05 0.05 0.15 0.25

1 /s Figure 1. Kinetics of the renal y-glutamyl transpeptidase- mediated hydrolysis (---) and transpeptidation (-) of 2-bromodiglutathion-S-ylhydroquinone (0 = hydrolysis; 0 = transpeptidation), 2-bromo-3-glutathion-S-ylhydroquinone (O), 2-bromo- 5-glutathion-S-ylhydroquinone (O) , and 2-bromo-6-glutathion- S-ylhydroquinone (A).

0 20 40 60 80 100

[CYSTEINE CONJUGATE] p.M

Figure 2. Concentration-dependent N-acetylation of (m) 2-bromo-5-cystein-S-ylhydroquinone, (0) 2-bromo-6-cystein-S-ylhydroquinone, (A) 2-bromo-3-cystein-S-ylhydroquinone, and (0) 2-bromodicystein-S-ylhydroquinone, catalyzed by renal microsomal cysteine conjugate N-acetylase. Values represent the mean f SD (n = 3-6). In some instances the SD is smaller than the size of the symbols.

116,62, and 50 pM, and the Vmm were 277,144 and 127 pmol/(unit-min), respectively. The rates of transpeptidation of the 2-Br-(monoGSyl)HQ’s were therefore ap- proximately 10-20-fold faster than the rate of hydrolysis. Interestingly, the kinetics of the hydrolysis and transpeptidation of 2-Br-(diGSyl)HQ were almost identical; the Km for both reactions (128 and 153 pM) was considerably higher than for the 2-Br-(monoGSyl)HQ conjugates whereas the Vmm was significantly lower [9 and 11 pmol/ (unit*min)]. Thus, the VmJKm values for the 2-Br- (monoGSy1)HQ conjugates were 7.5-fold higher (0.52 f 0.03 vs 0.069; hydrolysis) and 33-fold higher (2.43 f 0.06 vs 0.073; transpeptidation) than for 2-Br-(diGSyl)HQ.

Renal Microsomal N-Acetylation of 2-Bromocys- tein-S-ylhydroquinone T hioet hers. With the exception of 2-Br-3-(CYS)HQ, freshly prepared rat renal cortical microsomes catalyzed the N-acetylation of each of the cystein-S-yl conjugates in a concentration (25-100 pM)- dependent manner (Figure 2). The relative order of activity a t 100 pM was 2-Br-5-(CYS)HQ [ 18.7 f 0.94 nmol/ (mgmin)] < 2-Br-6-(CYS)HQ [13.4 f 0.88nmol/(mg.min)] << 2-Br-(diCYS)HQ [5.8 f 0.09 nmol/(mgmin)] < 2-Br-

Table 1. Effect of Ascorbic Acid on the N-Acetylation of 2-Bromocystein-S-ylhydroquinonesa

N-acetylation [nmol/ (mpmin)] substrate w/o ascorbate +ascorbate

13.3 f 0.2 18.0 f 0.4 (35) 2-bromo-5-cystein-S- ylhydroquinone

2-bromo-6-cystein-S- 8.6 f 0.4 11.7 f 0.2 (36) ylhydroquinone

2-bromodicystein-S- 3.1 f 0.03 6.5 f 0.1 (110) ylhydroquinone

a The N-acetylation of 2-bromocystein-S-ylhydroquinones (100 pM) was determined as described in the Materials and Methods, in the presence and absence of ascorbic acid (2 mM). The data are presented as the mean f SD of at least three separate experiments (n = 3-5). The values in parentheses represent the 5% increase in product formation [2-bromo(N-acetylcystein-S-yl)hydroquinones] in the presence of ascorbic acid.

2-Br-(tlicystein-.S-yI )hydroquinone

5 6 P 2 4

2

0 0 0 5 I 2

ASCORBATE ( m M ) Figure 3. Enhancement of the renal microsomal N-acetylation of 2-bromo-6-cystein-S-ylhydroquinone (100 pM) and 2-bromodicystein-S-ylhydroquinone (100 pM) by increasing concentrations of ascorbic acid. Values represent the mean f SD (n =

3-(CYS)HQ [3.2 f 0.15 nmol/(mg.min)]. Inclusion of ascorbic acid (2 mM) in the incubations increased the N-acetylation of 100 p M 2-Br-5-(CYS)HQ, 2-Br-6-(CYS)- HQ, and 2-Br-(diCYS)HQ by 35%, 36%, and l l O % , respectively (Table 1). The concentration-dependent effects of ascorbic acid on the N-acetylation of 2-Br- (diCYS)HQ and 2-Br-6-(CYS)HQ are shown in Figure 3. We assume that the inclusion of ascorbic acid (2 mM) was necessary to maximize the efficiency of the N-acetylation reaction by maintaining the substrates in the reduced form.

In Vitro N-Deacetylation of 2-Bromo(N-acetylcystein-S-yl) hydroquinone Thioet hers. 2-Br-3- (NAC)- HQ was not a substrate for the cytosolic N-deacetylase enzyme present in rat renal cortex, under the present experimental conditions. In contrast, 2-Br-(diNAC)HQ, 2-Br-5-(NAC)HQ, and 2-Br-6-(NAC)HQ were all deacetylated by the cytosolic N-deacetylase, in both a time- (linear to 30 min, data not shown) and concentration- dependent manner (Figure 4). The rate of the reaction varied in the order 2-Br-6-(NAC)HQ > 2-Br-5-(NAC)HQ >> 2-Br-(diNAC)HQ. At the highest (100 pM) concentration used, the relative rates of N-deacetylation were 304 f 40,280 f 32, and 196 f 20 pmol/(mgmin), respectively. N-Deacetylase activity toward 2-Br-6- (NAC)HQ (100 pM) was also observed in renal microsomes, a t a rate of about 5-10% of that seen with the cytosol [0.26 f 0.13 vs 5.2 f

3-6)

Determinants of Quinone Thioether-Mediated Toxicity Chem. Res. Toxicol., Vol. 7, No. 4, 1994 499

301 -

[A'-ACETYLCYSTEINE CONJUGATE] PM

Figure 4. Concentration-dependent N-deacetylation of (0) 2-bromo-6-(N-acetylcystein-S-yl)hydroquinone, (B) 2-bromo-5- (N-acetylcystein-S-yl)hydroquinone, and (e) 2-bromodi(N-acetylcystein-S-yl)hydroquinone, catalyzed by renal cortical cytosolic cysteine conjugate N-deacetylase. Values represent the mean * SD (n = 3-6). In some instances the SD is smaller than the size of the symbols.

0.08 nmol/(mg.20 min), respectively]. Whether this represents true microsomal activity, or residual cytosolic activity associated with the microsomes, is not known. Microsomes and cytosol prepared from rat liver displayed no activity toward any of the N-acetylcysteine conjugates under the experimental conditions employed.

For each of the compounds studied, the specific activity of the N-acetyltransferase clearly predominated over that of the N-deacetylase. The relative rates of these reaction were0.0341,0.023:1,0.015:l,and0:1 for the a-Br-(diNAC)- HQ:PBr-(diCYS)HQ, ~-BI--~-(NAC)HQ:~-B~-~-(CYS)HQ, 2-Br-5-(NAC)HQ:2-Br-5-(CYS)HQ, and 2-Br-3-(NAC)- HQ:2-Br-3-(CYS)HQ pairs, respectively. The order of N-deacetylationl N-acetylation cycling correlated exactly with the previously reported order of toxicity for both the 2-Br-(CYS)HQs and 2-Br-(NAC)HQs (28). The correlation (r2 = 0.99) between 2-Br-(NAC)HQ (100pmol/kg body wt)-mediated elevations in the urinary excretion of r-GT (data taken from Figure 4B in ref 28) and the corresponding N-deacetylationlN-acetylation ratio is shown in Figure 5.

Electrochemistry of 2-BrHQ-Thioethers. The hydrodynamic voltammograms of a-Br-(diGSyl)HQ, 2-Br- (diCYS)HQ, and 2-Br-(diNACYS)HQ are shown in Figure 6, with the Ell2 values for the corresponding monosub- stituted thioethers listed in Table 2. Under the experimental conditions employed in the present study, 2-Br- (diGSy1)HQ was more difficult to oxidize than 2-Br- (diNAC)HQ, whereas 2-Br-(diCYS)HQ was the most readily oxidized conjugate.

Discussion 2-Br-(diGSy1)HQ is a potent renal tubular toxicant that

requires metabolism by renal 7-GT for the expression of toxicity (14). Although a-Br-(diGSyl)HQ is a more potent nephrotoxicant than the 2-Br-(monoGSyl)HQs, in the present study we have shown that the latter conjugates are better substrates for renal y G T (Figure 1). A similar inverse relationship between the degree of GSH substitution of HQ and metabolism by 7-GT has been reported (25). It is therefore unlikely that differences in the rate of metabolism of the GSH conjugates by y G T contribute to their differential toxicity. In support of this view, the

U '

0.010 0.020 0.030

N-DEACETYLATION N- ACETYLATION

Figure 5. Correlation between the N-deacetylationlN-aceb ylation ratio and the relative nephrotoxicity of (stippled circlea) 2-bromo-6-(N-acetylcystein-S-y1) hydroquinone, (0) 2-bromo-S- (N-acetylcystein-S-yl)hydroquinone, and (0) 2-bromodi(N-acetylcystein-S-y1)hydroquinone. Toxicity was messed by mea- suring the urinary excretion of the proximal tubular brush border enzyme y-glutamyl transpeptidase following administration of each of the thioethers at a dose of 100 pmol/kg body wt (data taken from ref 28). The inset shows the same data (excretion of y-glutamyl transpeptidase) plotted on a logarithmic scale.

-0.2 0.0 0.2 0.4 0.6

Applied Potential (Volt) Figure 6. Hydrodynamic voltammograms of (0) 2-bromodiglutathion-S-ylhydroquinone (+286 mV), (B) 2-bromodicystein- S-ylhydroquinone (-66 mV), and (0) 2-bromodi(N-acetylcystein- S-y1)hydroquinone (+9 mV). Chromatographic conditions of the analysis are described in the Materials and Methods. The values in parentheses represent the Ell2 values.

Table 2. Half-Wave Oxidation Potentials of 2-Bromohydroquinone Monothioethers.

2-BrHQ-thioether Ella (mV) oxidation potential,

2-bromo-3-(N-acetylcystein-S-y1)- +79

2-bromo-6-(N-acetylcystein-S-y1)- +35

2-bromo-6-(N-acetylcyetein-S-y1)- +9

2-bromo-3-cystein-S- ylhydroquinone +77 2-bromo-6-cystein-S- ylhydroquinone +12

hydroquinone

hydroquinone

hydroquinone

2-bromo-6-cystein-S- ylhydroquinone +13 Potentials were determined by hydrodynamic voltammetry in

methanol/citrate at pH 4.0 with coulometric detection. capacity of total renal 7-GT (-4 X 105 unita/kidney) to metabolize 2-Br-(diGSyl)HQ appears to exceed the dose required to elicit toxicity (10-30 pmol/kg or 2-6 pmoll 200-g rat). The V,, values for the hydrolysis and transpeptidation of 2-Br-(diGSyl)HQ were almost identical, 9 and 11 pmol/(unit.min), respectively. A 200-g rat therefore has the capacity to metabolize between 7.2 and 8.8 pmol2-Br-(diGSyl)HQ/min. The corresponding values for the 2-Br-(monoGSyl)HQ conjugates range between 11

500 Chem. Res. Toxicol., Vol. 7, No. 4, 1994

pmollmin for the hydrolysis reaction and 102-222 pmoll min for transpeptidation. Thus, the activity of renal y G T is unlikely to be either the rate-limiting step in the metabolism of the GSH conjugates or a major factor governing differences in toxicity between 2-Br-(diGSyl)- HQ and the 2-Br-(monoGSyl)HQs. In support of this view, species differences in susceptibility to 2-Br-(diGSyl)- HQ-mediated nephrotoxicity do not correlate with differences in renal y G T activity (26). Therefore, factors other than renal T-GT are probably responsible for the differences in toxicity observed between mono- and multi- S-substituted quinones.

y G T catalyzes the first step in a series of reactions that usually results in mercapturic acid formation. Mercap- turates, the S-conjugates of N-acetylcysteine, are formed via the N-acetylation of cysteine thioethers by a requisite cysteine S-conjugate N-acetyltransferase (27). In vivo studies with male Sprague-Dawley rats showed that 2-Br- (CYS)HQ’s were more potent renal proximal tubular toxicants than the corresponding 2-Br-(NAC)HQ’s (28). Both 2-Br-(diCYS)HQ and 2-Br-(diNAC)HQ produce toxicity at doses lower than the corresponding monosub- stituted conjugates (28), which is consistent with the data obtained with the GSH conjugates. We therefore investigated whether variability in the relative rates of N- deacetylation and N-acetylation might contribute to the observed differences in toxicity. The relative nephrotoxic potency of the mercapturates correlated with the rate at which the conjugates underwent N-deacetylationlN- acetylation cycling (Figure 5) . Increasing the rate of N-deacetylation with respect to the reverse N-acetylation reaction increased the toxicity. Thus, 2-Br-54NAC)HQ and 2-Br-6-(NAC)HQ, which were the most toxic of the 2-Br-(monoNAC)HQ’s (28), were the most rapidly deacetylated (Figure 4), and 2-Br-3-(NAC)HQ, which caused little if any adverse effects following in vivo administration, was not a substrate for the N-deacetylase. a-Br-(diNAC)- HQ, the most potent of the mercapturates (281, was also effectively N-deacetylated. More importantly, however, the corresponding 2-Br-(diCYS)HQ was a relatively poor substrate for the N-acetyltransferase (Figure 21, which is consistent with the finding that the efficiency of the N-acetylation reaction is dependent upon the lipophilicity of the substrate (27). Consequently, the ratio of N- deacetylation to N-acetylation was highest for the 2-Br- (diNAC)HQ/2-Br-(diCYS)HQ pair (Figure 5). The results indicate that the N-deacetylationlN-acetylation ratio plays an important role in the toxicity of quinone thioethers.

Systemic administration of the cysteine conjugates, or their in situ formation via the activity of renal y G T on the corresponding GSH conjugates, results in their rapid delivery to renal proximal tubule cells, where three competing reactions may occur (Figure 7). The cysteine conjugates may either be N-acetylated (I), undergo cysteine conjugate @-lyase (29, 30) catalyzed elimination (111, or undergo oxidation (111) followed by arylation of tissue macromolecules (IV) andlor reactive oxygen generation (111). The data suggest that oxidation (and the conse- quences thereof) probably occurs much faster than either N-acetylation or @-elimination and that differences in the ease of oxidation of the conjugates provide the basis for the importance of the N-deacetylationlN-acetylation ratio. Thus, pretreatment of rats with (aminooxy)acetic acid (to inhibit cysteine conjugate @-lyase) did not prevent 2-Br- (diCYS)HQ-mediated nephrotoxicity and only partially

Monks et al.

OH nu

0;- O 2 4 \ 1 e-

H 20 NHi h NH: b H

Figure 7. Renal disposition of 2-bromocystein-5’-ylhydroquinone%. Following either systemic administration or in situ formation via y-glutamyl transpeptidase-mediated metabolism of the corresponding GSH conjugate, three major metabolic pathways may occur. The cysteine conjugate may be N-acetylated to the mercapturic acid (I) or undergo @-elimination to the thiophenol (11). However, the data suggest that oxidation (111) is probably the major route of metabolism, with either the concomitant generation of reactive oxygen species (111) and/or arylation of tissue macromolecules (IV). Oxidative cyclization and 1,4- benzothiazine formation (V) probably constitutes a minor metabolic pathway. The ability of the protein adduct to undergo further oxidation, leading to either protein cross-linking and/or reactive oxygen generation, requires further investigation.

protected against 2-Br-(monoCYS)HQ nephrotoxicity (28). Consistent with these findings, the efficiency of the N-acetylation reaction was substantially decreased in the absence of ascorbic acid (Table 1, Figure 3), and the omission of ascorbic acid had a more dramatic effect on the N-acetylation of 2-Br-(diCYS)HQ than on the N- acetylation of 2-Br-(monoCYS)HQ’s. This indicates that a greater fraction of 2-Br-(monoCYS)HQ will be available as a substrate for @-lyase than 2-Br-(diCYS)HQ, inasmuch as 2-Br-(diCYS)HQ (Figure 6) has a lower oxidation potential than the 2-Br-(monoCYS)HQ’s (Table 1) and should be more readily oxidized to the reactive quinone. Moreover, intramolecular cyclization and 1,Cbenzothi- azine formation (31) (Figure 7, V) probably constitutes a minor pathway of 2-bromocystein-S-yl-1,4-benzoquinone disposition, since this reaction eliminates the reactive quinone function from the molecule and would therefore be a pathway of detoxication. The reason why (aminooxy)- acetic acid blocks the toxicity of systemically administered 2-Br-(diNAC)HQ (28) is unclear, but may be related to differences in the site of delivery of the conjugate to renal proximal tubule cells (see Figure 1 in ref 1). Thus, 2-Br- (diNAC)HQ is transported into renal proximal tubule cells via the probenecid-sensitive organic anion carrier (28) located on the basal-lateral membrane, whereas 2-Br- (diCYS)HQ is probably transported into renal cells via amino acid carriers located on both the apical and basal- lateral membranes.

The exponential relationship between the N-deacetyl- ationlN-acetylation ratio and nephrotoxicity (Figure 5) is similar to the dose-response relationship previously reported for both 2-Br-(diGSyl)HQ (12) and 2,3,5-(triG- Sy1)HQ (21). The nonlinear dose-response relationship suggests the presence of a factor(s) that at low doses protects the kidney from toxicity, but becomes depleted a t a certain threshold dose. Since the specific activity of

Determinants of Quinone Thioether-Mediated Toxicity

the N-acetyltransferase [nmol/(mg.min)l (Figure 2) is between 1 and 2 orders of magnitude greater than that of the N-deacetylase [pmol/(mg.min)l (Figure 4), the threshold effect may be due to depletion of either the acetyl- CoA cofactor and/or its precursors (fatty acids, HSCoA or ATP). This suggestion is consistent with the conclusions reached by Commandeur et al. (32, 33). These authors concluded that the rapid N-acetylation of S-(1,1,2,2,- tetrafluorethy1)-L-cysteine, followed by deacetylation and subsequent reacetylation, resulted in levels of acetyl-coA that became rate-limiting (32). Thus, as the fraction of S- (1,1,2,2,-tetrafluorethyl)-~-cysteine available for detoxication by N-acetylation decreased, the fraction available for bioactivation via cysteine conjugate @-lyase increased. In addition, the relative toxicity of N-acetyl-S-(1,1,2,2- tetrafluorethy1)-L-cysteine, N-acetyl-S-(l-chloro-l,2,2-tri- fluoroethy1)-L-cysteine, N-acetyl-S-(l,l-dichloro-2,2-dif- luorethy1)-L-cysteine, and N-acetyl-S-(1,l-dibromo-2,2- difluorethy1)-L-cysteine in rat renal slices (34) correlated with their relative rates of deacetylation (33).

In summary, the nephrotoxicity of 2-BrHQ-thioether conjugates is determined by a combination of enzymatic (N-acetylation/N-deacettylation) and redox factors. Dif- ferences exhibited by the mono-S- and di-S-substituted thioethers correlated with the relative rate at which they underwent N-deacetylationl N-acetylation cycling (Figure 5) and by the ease with which they undergo oxidation to the corresponding quinone thioether (Figure 6 and Table 2).

Acknowledgment. This work was supported in part by U.S. Public Health Service Award ES 04662 from the National Institute of Environmental Health Sciences (T.J.M.).

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Fowler, L. M., Moore, R. D., Foster, J. R., and Lock, E. A. (1991) Nephrotoxicity of 4-aminophenol glutathione conjugate. Hum. Exp. Toxicol. 10, 451-459. Klos, C., Koob, M., Kramer, C., and Dekant, W. (1992) p- Aminophenol nephrotoxicity: Biosynthesis of toxic glutathione conjugates. Toxicol. Appl. Pharmacol. 115, 98-106. Mertens, J. J. W. M., Temmink, J. H. M., van Bladeren, P. J., Jones, T. W., Lo, H.-H., Lau, S. S., and Monks, T. J. (1991) Inhibition of y-glutamyl transferase potentiates the nephrotoxicity of glutathione conjugated chlorohydroquinones. Toricol. Appl. Pharmacol. 110, 45-60. Lau, S. S., Jones, T. W., Highet, R. J., Hill, B. A,, and Monks, T. J. (1990) Differences in the localization and extent of the renal proximal tubular necrosis caused by mercapturic acid and glutathione conjugates of menadione and l,4-naphthoquinone. Toxicol. Appl. Pharmacol. 104, 334-350. Kari, F. W. (1989) Toxicology and carcinogenesis studies of hydroquinoine in F344/N rata and B6C3F1 mice (gavage studies). National Toxicology Program Technical Report No. 366, pp 1-86, US. Department of Health and Human Services, USPHS, NIH. Shibata, M.-A., Hirose, M., Tanaka, H., Asakawa, E., Shirai, T., and Ito, N. (1991) Induction of renal cell tumors in rata and mice, and enhancement of hepatocellular tumor development in mice after long-term hydroquinone treatment. Jpn. J. Cancer Res. 82,1211- 1219. Hill, B. A,, Kleiner, H. E., Ryan, E. A,, Dulik, D. M., Monks, T. J., and Lau, S. S.(1993) Identificationof multi-S-substituted conjugates of hydroquinone by HPLC-coulometric electrode array analysis and mass spectroscopy. Chem. Res. Toxicol. 6, 459-469. Lau, S. S., Hill,B. A.,Highet, R. J., andMonks,T. J. (1988) Sequential oxidation and glutathione addition to 1,4-benzoquinone: Correlation of toxicity with increased glutathione substitution. Mol. Pharmacol. 34,829-836. Larsson, R., Boutin, J., and Moldeus, P. (1988) Peroxidase-catalysed metabolic activation of xenobiotics. In Metabolism of Xenobiotics (Gorrod, J. W., Oelschlager, H., and Caldwell, J., Eds.) pp 43-50, Taylor and Francis, New York. Canales, P. L., Kleiner, H. E., Monks, T. J., and Lau, S. S. (1993) Formation of 8-hydroxydeoxyguanosine by quinol-thioethers. Toxi- cologist 13, 202 (Abstract). Hill, B. A., Lo, H.-H., Monks, T. J., and Leu, S. S. (1991) The role of y-glutamyl transpeptidase in hydroquinone-glutathione conjugate mediated nephrotoxicity. In Biological Reactiue Intermediates IV. Molecular and CellularEffectsand theirlmpact on Human Health (Witmer, C. M., Snyder, R. R., Jollow, D. J., Kalf, G. F., Kocsis, J. J., and Sipes, I. G., Eds.) pp 749-751, Plenum Press, New York. Lau, S. S., Jones, T. W., Sioco, R., Hill, B. A., Pinon, R. K., and Monks, T. J. (1990) Species differences in renal y-glutamyl transpeptidase activity do not correlate with susceptibility to 2- bromo- (diglutathion-S-yl) hydroquinone mediated nephrotoxicity.

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(32) Commandeur, J. N. M., De Kanter, F. J. J., and Vermeulen, N. P. E. (1989) Bioactivation of the cysteine-S-conjugate and mercapturic acid of tetrafluoroethylene to acylating reactive intermediates in the rat: Dependence of activation and deactivation activities on acetyl coenzyme A availability. Mol. Phurmucol. 36,664-663.

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Documents

Oxidation and Acetylation as Determinants of 2-Bromocystein-S-ylhydroquinone-Mediated Nephrotoxicity