Covalent Protein Adducts of Hydroquinone in ... - ACS Publications

Chem. Res. Toxicol. 2000, 13, 861-872

861

Covalent Protein Adducts of Hydroquinone in Tissues from Rats: Quantitation of Sulfhydryl-Bound Forms following Single Gavage or Intraperitoneal Administration or Repetitive Gavage Administration† 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 current studies were conducted to investigate the degree and type of protein binding of hydroquinone (HQ) in the rat following single oral or intraperitoneal (ip) or repeated oral administrations. Male or female F-344 rats or male SD rats received a single dose of HQ at 0, 25, 50, or 100 mg/kg by either gavage or ip injection (SD rats only). In addition, male or female F-344 or male SD rats received HQ by gavage for 6 weeks (5 days/week) at 0, 25, or 50 mg/ kg/day. Sulfhydryl-bound HQ was quantitated in protein from blood, kidneys, livers, or spleens 24 h after treatment using an alkaline permethylation procedure. The amount of total protein-S adducts increased with increasing dose in all the tissues that were assayed. Female rats had higher levels of adducts in blood, livers, and kidneys than did male rats when they were treated orally. Male F-344 rats treated orally had elevated levels of adducts in these same tissues compared to SD rats treated orally. For all genders and strains of rats and for all treatment regimens, mono-adducts predominated in livers (>72% of total). In the kidneys, tri- and tetrasubstituted adducts predominated with the summation accounting for >60% of the total. Ip administration of HQ resulted in significantly elevated levels of adducts in all the tissues that were examined, with the greatest increases seen for protein from blood and spleens. Levels of protein-S adducts of HQ in rat kidney following a single gavage administration correlated well with previously published differences in acute HQ nephrotoxicity in rats (female F-344 rat > male F-344 rat > male SD rat). Elevated levels of HQ protein-S adducts following repeated gavage administration did not correlate to measurable clinical signs of nephrotoxicity. Evidence is presented suggesting a possible role for the prostaglandin H synthase complex in the metabolic activation of HQ. In addition, protein arylation alone cannot account for the greater sensitivity of male F-344 rats toward chronic administration of HQ. The sensitivity of male F-344 rats to HQ is likely due to other factors, including the incidence and severity of chronic progressive nephropathy.

Introduction The Fischer 344 (F-344)1 strain of rat displays a weak nephrotoxic response to HQ following either single or repeated oral treatment, resulting in renal tubular cell degeneration and mild enzymuria and glucosuria (1, 2). Additionally, the female F-344 rat is more sensitive to the acute nephrotoxicity of HQ than is the male, whereas SD rats are insensitive (1). In a recent publication from this laboratory (1), oral administration of HQ was shown to produce enzymuria and glucosuria following single acute doses in male F-344 rats (at 400 mg/kg) and in female F-344 rats (at doses of 200 and 400 mg/kg). These effects were more pronounced in female F-344 rats. Neither male nor female SD rats showed evidence of nephrotoxicity due to HQ administration at 400 mg/kg by gavage. In male F-344 rats given 200 mg/kg HQ for 13 weeks, moderate to marked renal tubular cell degen† 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, 1,4-benzoquinone; F-344, Fischer 344 strain of albino rat; SD, Sprague-Dawley strain of albino rat; CPN, chronic progressive nephropathy.

eration was observed, while in females, lesions were minimal to mild (2). Similarly, English et al. (3) reported an increased sensitivity of the male F-344 rat for nephrotoxocity following HQ exposures for 6 weeks (3). The mechanisms underlying this increased sensitivity are not known, but a deficiency in HQ-cysteine conjugate Nacetylation activity in the kidney may contribute to enhanced toxicity in the male F-344 rat (4). Also, the incidence and severity of chronic progressive nephropathy (CPN) in the male F-344 may play a significant role in enhancing nephrotoxicity following chronic exposures. In a recent histopathological review by Hard et al. (5), it was proposed that an enhancement of the severity of CPN, coupled with stimulation of tubule proliferation, may lead to the observed increase in the extent of HQrelated kidney adenoma formation. The role of CPN in enhancing HQ-induced effects may be pivotal to induction of renal adenomas since older F-344 rats, without significant CPN, are less sensitive to HQ than are young rats (6). A variety of structurally related chemicals, including acetaminophen (7, 8), p-aminophenol (9), and 2-bromoHQ (10), are nephrotoxic. Metabolic activation through for-

10.1021/tx000038p CCC: $19.00 © 2000 American Chemical Society Published on Web 08/11/2000

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mation of a glutathione conjugate is apparently required for this activity. Thus, conversion to a reactive arylating agent and subsequent protein binding may lead to the observed nephrotoxic responses (11). In the case of HQ, the triglutathionyl conjugate [2,3,5-(triglutathion-S-yl)HQ] has been shown to be the most nephrotoxic conjugate of HQ, producing elevated bood urea nitrogen levels in animals treated ip with the conjugate at dose levels as low as 5 µmol/kg (12), and in addition, this conjugate is bound significantly to kidney protein when administered directly to the kidney in an in vivo perfused kidney assay (13). Further conversion of the conjugate within the kidney is required for this response to be manifested. Thus, pretreatment with AT-125, an irreversible inhibitor of γ-glutamyl transpeptidase, completely eliminates the nephrotoxicity of this conjugate when administered iv (12). It has been recognized for many years that the extent of covalent binding alone is not always predictive of the toxicity of a given compound or reactive metabolite (14). As in the case of bromobenzene and acetaminophen hepatotoxicity, a threshold dose must be achieved before tissue damage is observed (15). Significant amounts of trans-stilbene (a weak synthetic estrogen) are bound to liver and kidney protein up to doses which deplete glutathione levels; however, no significant liver or kidney histopathological changes are seen (16). Acetyl-m-aminophenol, a regioisomer of acetyl-p-aminophenol (APAP) possessing similar analgesic and antipyretic properties, was approximately 10 times less toxic to cultured mouse hepatocytes despite a greater extent of protein binding in this system (17). Clearly, the nature of the protein-S adducts that are formed can be critical to the resultant tissue response. The current studies were undertaken to determine the extent and nature of covalent protein binding of HQ to various tissues in male and female F-344 and male SD rats following either a single oral or intraperitoneal administration or repeated gavage administration. Due to the known chemical instability of quinone protein-S adducts, an alkaline “permethylation” procedure described by Slaughter and Hanzlik (18) was employed for the quantitation of protein covalent adducts. In this procedure, basic hydrolysis of tissue proteins results in a chemical fragmentation of sulfhydryl-bound HQ adducts which, in the presence of excess methyl iodide, are rapidly converted to a number of chemically stable methylthio derivatives. As described in the preceding article (19), a GC/MS procedure was developed which allowed for the simultaneous quantitation of all six of the possible sulfhydryl-substituted HQ products. Also during the current studies, the possibility that HQ treatment alters renal prostaglandin synthesis was investigated. HQ is capable of modulating prostaglandin synthesis in polymorphonuclear leukocytes in vitro (20). Prostaglandins produced in the kidney regulate renal hemodynamics, and the effects of HQ and its glutathione conjugates on kidney tissue may be secondary to stimulation or inhibition of prostaglandin synthesis. The effect of repeated gavage administration of HQ on prostaglandin synthesis was determined by measurement of thromboxane B2 and 6-keto-prostaglandin F1R levels in urine. The results of these analyses are discussed in terms of the known subchronic and chronic toxicities of HQ.

Boatman et al.

Experimental Procedures Materials and Methods. Hydroquinone was obtained from the Eastman Kodak Co. (Rochester, NY). The chemical purities of all dose preparations were >98% as determined by HPLC. The glutathione conjugates of hydroquinone were prepared as standards by a modification of the procedure of Lau et al. (12) and purified by preparative HPLC. The characterization of the glutathione conjugates and the details of the permethylation procedure used for analysis are described in the preceding paper (19). Methyl iodide and resorcinol were obtained from Aldrich Chemical Co. (Milwaukee, WI) and were reagent grade. Pentane (Aldrich Chemical Co.) was HPLC grade. Heptane was obtained from EM Sciences, Inc. (Cherry Hill, NJ) and was rated suitable for residue analysis. All other chemicals were reagent grade unless otherwise noted. Stock solutions of HQ were prepared in degassed, distilled water or degassed, sterile saline at concentrations not exceeding 50 mg/mL. The concentration of HQ in treatment solutions was determined by HPLC analysis immediately prior to dose administration. Animals. Male and female F-344 [CDF(F-344)/Crl BR] and male SD [Cr1:CD(SD)BR] rats were purchased from Charles River Kingston (Stone Ridge, NY). These strains of rats have been used extensively for toxicity and carcinogenicity studies and have been used previously for toxicity studies with HQ. The rats were from 9 to 12 weeks of age at the time of treatment. All test animals were held in isolation for at least 5 days prior to use. Animals were examined during this period, and any that appeared to be in poor condition were removed from the study. Animals were housed in wire-mesh, stainless steel cages within an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility. Room lighting followed a 12 h light/dark cycle, without twilight. Room temperatures and relative humidities were maintained at 23 ( 3 °C and 30-70%, respectively. Ventilation was provided at a rate of 10-15 room air changes per hour. Animals were fed certified rodent diet (Agway Prolab RMH 3000 or Agway Prolab RMH 3200 meal) ad libitum. Domestic tap water (Monroe County Water Authority, Monroe County, NY) was available ad libitum. Neither the feed nor the water contained contaminants at concentrations that might be expected to interfere with the interpretation of the study. All animals were identified individually using uniquely numbered ear tags. Test Article Administration. For single administration studies, rats were given HQ by oral gavage in degassed, distilled water or by intraperitoneal injection in degassed, sterile saline at the following doses: 0 (vehicle control), 25, 50, or 100 mg/kg. In 6 week studies, HQ was given daily (5 days/week) by oral gavage in degassed, distilled water at the following doses: 0 (vehicle control), 25, or 50 mg/kg. Doses were determined for daily administrations on the basis of body weights obtained on the first day of exposure (Monday) of each week. Constant administration volumes of 5 and 2.5 mL/kg were employed for oral gavage and ip treatment, respectively. Tissue. At the termination of each study, rats were anesthetized (CO2) and exsanguinated via the inferior vena cava. Blood was collected in tubes containing EDTA. The livers, kidneys, and spleens were removed and placed immediately into plastic bags or glass bottles stored over ice. Tissue samples which were not further processed on the day of collection were stored frozen at -70 °C until they were analyzed. Immediately following the administration of the last dose in 6 week studies, all animals were transferred to Nalgene (Nalge Co., Rochester, NY) metabolism cages, and urine specimens (24 h) were collected on ice and stored refrigerated until analysis. Preparation of Protein Samples, Permethylation, and Analysis. The details of these procedures are described fully in the preceding paper (19). The terminology used for the various HQ-sulfhydryl protein adducts is that given in Figure 2 of the preceding article (19). Urine Analyses. Prior to analysis, urine samples were centrifuged for 10 min at 1000g. The volume of each urine

HQ-Protein Adducts following Administration to Rats

Chem. Res. Toxicol., Vol. 13, No. 9, 2000 863 (Indianapolis, IN); γ-GT, using Dri-STAT reagent from Beckman; glucose, using glucose determination reagents (GDH endpoint) from Seradyn Inc. (Indianapolis, IN); and creatinine, using packaged reagents from Roche Diagnostic Systems, Inc. Thromboxane B2 and 6-keto-prostaglandin F1R were assessed in diluted urine (1:10 v:v) using Biotrak enzyme immunoassay kits from Amersham (kits RPN 221 and RPN 220, Amersham Corp.). Statistical Analyses. The results for individual and total protein-S adduct levels were analyzed using descriptive statistics, plots, tests for normality and homogeneity of variance, and Dunnet’s t test for mean comparisons. All statistical analyses were two-tailed tests of significance using an R risk of 0.05. Statistical calculations were performed using the computer program SAS (version 6, SAS Institute, Cary, NC).

Results

Figure 1. Levels of tetrasubstituted adducts of HQ in protein from livers, blood, and kidneys from groups of four male or female F-344 or male SD rats treated once orally or ip with HQ at 50 mg/kg. Error bars represent 1 SD.

Figure 2. Levels of tetrasubstituted adducts of HQ in protein from livers, blood, and kidneys from groups of five male or female F-344 or male SD rats treated orally with HQ at 50 mg/ kg for 6 weeks (5 days/week). Error bars represent 1 SD. sample was recorded. For urinary alanine aminopeptidase (AAP) analysis, ethylene glycol was added to the supernatants from the centrifugation (0.75 mL of glycol to 1.75 mL of urine), and the supernatants were stored at -20 °C prior to analysis (21). Urine samples to be analyzed for AAP were purified by gel filtration on Sephadex G-25 M (PD10) columns (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ) using a methodology modified from that of Berscheid et al. (21). A COBAS BIO centrifugal analyzer (Roche Diagnostic Systems, Inc., Nutley, NJ) was employed for the following assays of urine: alanine aminopeptidase (AAP), by the procedure of Mattenheimer et al. (22); N-acetyl-β-D-glucosaminidase (NAG), using NAGase test kit reagents from Boehringer Mannheim Biochemicals, Inc.

Single-Dose Administration. (1) Oral. In a study employing a single oral dose of HQ, groups of four male or female F-344 rats or male SD rats were treated by gavage with HQ at 0 (vehicle control), 25, 50, or 100 mg/ kg and the levels of protein-S adducts of HQ measured in blood, kidneys, livers, and spleens 24 h after treatment. Total amounts of protein-S-bound HQ were higher in livers and kidneys than in blood or spleens at all dose levels (see Table 1). A clear dose-response (increasing adduct levels with increasing dose) was seen for all tissue protein-S adducts with the exception of the spleen protein from male F-344 rats. Greater amounts of HQ were bound in livers and kidneys from female F-344 rats than from male rats (Table 1). Values for liver adducts in females ranged from 46.8 (untreated) to 1003 pmol/ mg (100 mg/kg), and values for kidney adducts in females ranged from 24.8 (untreated) to 304 pmol/mg (100 mg/ kg). Levels of adducts found in livers and kidneys of male F-344 rats were nominally higher than for SD rats (Table 1). Values for liver adducts in male F-344 rats ranged from 19.9 (untreated) to 478 pmol/mg (100 mg/kg) versus similar values of 11.1 (untreated) to 344 pmol/mg (100 mg/kg) for SD rats; values for kidney adducts in male F-344 rats ranged from 7.8 (untreated) to 202 pmol/mg (100 mg/kg) versus similar values of 19.3 (untreated) to 162 pmol/mg (100 mg/kg) for SD rats. Lower concentrations of total protein-S adducts were present in protein from whole blood or spleens following oral administration of HQ than were present in kidney or liver (see Table 1). In blood, female F-344 rats showed the highest levels of protein-S adducts with values for total protein-S adducts ranging from 3.8 (untreated) to 62.0 pmol/mg (100 mg/kg). Levels of adducts found in blood from male SD rats were nominally higher than for F-344 rats. In male SD rats, values ranged from 4.2 (untreated) to 47.1 pmol/mg (100 mg/kg) versus similar values of 2.9 (untreated) to 30.2 pmol/mg (100 mg/kg) for F-344 rats. In the case of spleen protein, female F-344 rats exhibited higher levels of protein-S adducts in this tissue than did male SD rats treated orally at equivalent levels. Values for adducts in spleens from female rats ranged from 7.9 (untreated) to 119 pmol/mg (100 mg/kg). Male SD rats had the lowest levels of adducts to spleen protein with values ranging from none detected (untreated) to 24.2 pmol/mg (100 mg/kg). Values for spleen adducts in male F-344 rats ranged from 54.1 (untreated) to 204 pmol/mg (50 mg/kg) with no clearly evident doseresponse relationship. Assays of spleen proteins from male F-344 rats were compromised by high levels of

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Table 1. Levels of Total Protein-S Adducts in Blood, Kidneys, Livers, and Spleens from Groups of Four Male or Female F-344 or Male SD Rats Treated Orally or ip (single dose) with HQ at 0 (control), 25, 50, or 100 mg/kg total protein adduct level (pmol/mg of protein) ((SD) blood male F-344, oral 0 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg female F-344, oral 0 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg male SD, oral 0 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg male SD, ip 0 mg/kg 25 mg/kg 50 mg/kg 100 mg/kg

kidney

liver

spleen

2.94 ( 0.16 3.06 ( 2.16 8.11 ( 4.04 30.2 ( 7.31b

7.84 ( 2.00 44.0 ( 15.2b 87.8 ( 40.1b 202 ( 26.8b

19.9 ( 6.04 90.6 ( 34.0 165 ( 20.8b 478 ( 206b,f

54.1 ( 13.8 53.2 ( 32.0 37.2 ( 5.3 72.3 ( 22.2

3.81 ( 2.11 11.9 ( 6.04 32.7 ( 11.6b,e 62.0 ( 19.5b,e

24.8 ( 3.72 89.5 ( 20.6b 153 ( 38.1b 304 ( 36.4b,c

46.8 ( 16.8 240 ( 41.0b,c 396 ( 63.8b,c 1003 ( 219b,c

7.95 ( 9.26 39.3 ( 41.0b 43.7 ( 14.2b 119 ( 43.2b

4.23 ( 1.42 4.88 ( 1.20 15.0 ( 6.38b 47.1 ( 31.8b

19.3 ( 2.01 36.8 ( 15.1 103 ( 12.8b 162 ( 39.1b

11.1 ( 2.66 91.4 ( 11.3 210 ( 48.2b 344 ( 73.8b

NDa 4.55 ( 3.94b 18.9 ( 6.72b 24.2 ( 7.79b

4.64 ( 1.34 49.9 ( 7.63b,h 112 ( 23.5b,d 348 ( 96.0b,d

23.9 ( 3.99 129 ( 29.6b 341 ( 52.3b,d 792 ( 143b,d

10.2 ( 2.23 79.6 ( 14.2 301 ( 120b,g 644 ( 156b

5.53 ( 1.92 53.7 ( 4.87b 140 ( 6.16b 296 ( 26.0b,d

a ND, none detected. Below the detection limit (approximately 1-2 pmol/mg of protein) of the analytical procedure that was used. Value statistically significantly different from the control value (p e 0.05). c The value for females was statistically significantly different from that for males (oral) at this dose level (p e 0.05). d SD rats treated ip were statistically significantly different from all other groups at this dose level (p e 0.05). e The value for females was statistically significantly different from that for male F-344 rats at this dose (oral, p e 0.05). f The value for male F-344 rats was statistically significantly different from that for male SD rats at this dose level (oral, p e 0.05). g The value for male SD rats treated ip was statistically significantly different from that for male F-344 rats (p e 0.05). h The value for male SD rats treated ip was statistically significantly different from that for male SD rats treated orally at this dose level (p e 0.05).

b

interfering substances chromatographing in the region containing the di-adducts, resulting in inflated levels of these adducts. (2) Ip. Male SD rats treated ip with HQ showed significantly increased levels of total HQ protein-S adducts in all tissues that were assayed compared with similar values from oral treatment. The largest increase was seen for the spleen protein (see Table 1). The amount of total adducts in spleens increased approximately 7-12fold with values ranging from 5.5 (untreated) to 296 pmol/ mg (100 mg/kg). The amount of total adducts in blood increased 7-10-fold and in kidneys increased 3-5-fold over comparable values from oral treatment; values in blood ranged from 4.6 (untreated) to 348 pmol/mg (100 mg/kg), and values in kidney ranged from 23.9 (untreated) to 792 pmol/mg (100 mg/kg). Levels of protein-S adducts in liver after ip treatment were approximately 1-1.9 times the values obtained after oral treatment with values ranging from 10.2 (untreated) to 644 pmol/mg (100 mg/kg). The relative proportions of individual HQ adducts (mono, di, tri, or tetra) following either a single oral or ip administration varied as a function of tissue (see Tables 2-5). The mono-adduct accounted for the majority (>76%) of the protein-S-bound HQ in liver samples (Table 2). In kidneys (Table 3), the sum of the tri- and tetra-adducts accounted for the majority (>60%) of the protein-S-bound HQ. In spleens (male F-344 excluded), the mono-adduct was still the predominant form accounting for >58% of the total adducts (Table 4). In blood (Table 5), a distribution similar to that of spleen was observed. For a given tissue (with the exception of the results for spleens from male F-344 rats), the relative proportions of the various adducts did not appear to vary as a function of either gender, strain of rat, or route of administration.

Six Week Gavage Administration. (1) Adducts to Liver and Kidney Protein. Separate groups of five male or female F-344 or male SD rats were treated orally for 6 weeks (5 days/week) at 0, 25, or 50 mg/kg/day and HQ protein-S adducts levels measured in tissues collected 24 h after final treatment. Amounts of total protein-S-bound HQ were higher in livers and kidneys than in blood or spleens at all dose levels (Table 6). Greater amounts of HQ were bound in livers and kidneys from female F-344 rats than from males; values for the amounts of total liver adducts in females ranged from 74.0 (untreated) to 904 pmol/mg (50 mg/kg), and values for the amounts of total kidney adducts in females ranged from 17.9 (untreated) to 308 pmol/mg (50 mg/kg). Levels of total adducts found in livers and kidneys of male F-344 rats were nominally higher than those in SD rats; values for liver adducts in male F-344 rats ranged from 21.4 (untreated) to 472 pmol/mg (50 mg/kg) versus similar values from 13.7 (untreated) to 449 pmol/mg (50 mg/kg) for SD rats, and values for total kidney adducts in male F-344 rats ranged from 9.2 (untreated) to 170 pmol/mg (50 mg/kg) versus values from 9.1 (untreated) to 140 pmol/mg (50 mg/kg) for SD rats. (2) Adducts to Blood and Spleen Protein. Lower concentrations of total adducts were present in protein from whole blood or spleens following a 6 week oral administration of HQ than were present in kidney or liver (see Table 6). In blood, female F-344 rats showed the highest levels of total protein-S adducts with values ranging from 10.7 (untreated) to 230 pmol/mg (50 mg/ kg). Levels of adducts found in blood from male F-344 rats were similar to those from SD rats; in male F-344 rats, values ranged from 10.3 (untreated) to 130 pmol/ mg (50 mg/kg) versus similar values from 11.8 (untreated) to 128 pmol/mg (50 mg/kg) for SD rats. In the case of spleen protein, female F-344 rats again exhibited

0 mg/kg

135 ( 15.9 4.67 ( 1.61 7.34 ( 1.49 3.63 ( 1.61 3.61 ( 0.871 11.6 ( 2.02

The error represents (1 SD.

71.5 ( 29.5 3.20 ( 1.13 4.00 ( 1.77 2.10 ( 1.20 2.45 ( 0.643 7.34 ( 2.22

50 mg/kg

male F-344, oral

25 mg/kg 401 ( 192 10.3 ( 3.13 17.9 ( 8.53 11.7 ( 5.37 11.2 ( 3.88 25.9 ( 6.99

100 mg/kg 27.6 ( 10.9 1.85 ( 0.825 3.10 ( 1.30 1.43 ( 0.944 3.72 ( 2.04 9.07 ( 1.67

0 mg/kg 184 ( 32.6 7.44 ( 1.67 13.7 ( 3.68 4.85 ( 4.19 12.3 ( 2.91 18.2 ( 4.42

319 ( 54.8 10.6 ( 1.09 19.6 ( 2.81 10.3 ( 11.3 13.3 ( 8.41 23.6 ( 2.62

50 mg/kg

female F-344, oral 25 mg/kg

0 mg/kg

848 ( 192 8.77 ( 1.04 21.6 ( 4.78 ND 50.4 ( 9.52 ND 9.03 ( 3.30 1.03 ( 0.702 31.4 ( 6.82 0.478 ( 0.249 42.8 ( 6.42 0.866 ( 1.02

100 mg/kg 73.7 ( 9.40 1.77 ( 0.108 3.66 ( 0.467 2.55 ( 1.46 2.92 ( 0.397 6.87 ( 0.845

177 ( 43.6 3.40 ( 0.326 7.51 ( 1.15 5.60 ( 2.32 4.64 ( 0.270 12.8 ( 1.85

50 mg/kg

male SD, oral 25 mg/kg

0 mg/kg

288 ( 67.0 8.19 ( 0.849 5.73 ( 0.928 ND 12.6 ( 1.87 ND 9.12 ( 3.06 0.833 ( 0.962 7.25 ( 1.10 0.765 ( 0.059 21.2 ( 4.41 0.392 ( 0.784

100 mg/kg

66.5 ( 12.2 1.79 ( 0.395 3.61 ( 1.29 1.13 ( 1.23 2.02 ( 0.826 4.53 ( 2.01

249 ( 96.1 5.79 ( 2.47 12.5 ( 5.80 6.81 ( 5.21 5.91 ( 3.86 21.3( 7.35

50 mg/kg

male SD, ip 25 mg/kg

549 ( 139 11.4 ( 2.69 24.7 ( 6.18 13.1 ( 4.68 12.9 ( 3.68 32.5 ( 6.23

100 mg/kg

6.24 ( 2.77 8.33 ( 4.27 1.54 ( 1.88 5.56 ( 2.05 19.3 ( 8.40 46.8 ( 23.7

50 mg/kg


3.51 ( 1.84 4.51 ( 3.56 ND 2.21 ( 2.73 9.93 ( 3.53 23.9 ( 7.32

2.66 ( 1.01 2.58 ( 1.75 ND ND 1.58 ( 0.706 1.02 ( 2.04

mono 2,3-di 2,5-di 2,6-di tri tetra

a

25 mg/kg

0 mg/kg

adduct

male F-344, oral 0 mg/kg

50 mg/kg


28.0 ( 10.2 4.24 ( 0.775 16.6 ( 5.47 36.5 ( 12.0 10.7 ( 4.67 7.31 ( 8.86 10.4 ( 4.24 10.3 ( 4.08 7.03 ( 2.05 ND 0.673 ( 1.35 6.03 ( 2.37 10.8 ( 1.13 0.729 ( 1.46 7.40 ( 1.37 10.7 ( 1.88 44.0 ( 3.52 4.43 ( 1.00 16.9 ( 3.11 26.1 ( 5.38 102 ( 8.05 8.11 ( 6.07 37.6 ( 8.06 63.5 ( 14.1

100 mg/kg

76.1 ( 10.8 16.0 ( 2.21 10.5 ( 2.39 16.0 ( 1.61 54.0 ( 6.84 132 ( 16.4

100 mg/kg

2.56 ( 0.974 6.28 ( 1.42 ND ND 1.36 ( 0.319 9.06 ( 0.990

0 mg/kg

3.30 ( 1.06 9.39 ( 5.20 ND ND 7.48 ( 2.29 16.7 ( 7.03

25 mg/kg

13.4 ( 5.27 11.5 ( 3.27 4.24 ( 2.87 4.81 ( 1.77 24.7 ( 4.03 44.4 ( 6.30

50 mg/kg

male SD, oral

27.4 ( 13.7 13.9 ( 5.01 8.09 ( 1.73 5.44 ( 2.90 36.1 ( 10.2 71.5 ( 21.3

100 mg/kg

4.65 ( 0.740 9.55 ( 3.21 ND ND 2.00 ( 0.557 7.66 ( 1.93

0 mg/kg

26.0 ( 1.78 9.07 ( 1.60 4.66 ( 1.49 5.23 ( 1.15 24.0 ( 6.72 59.7 ( 22.5

25 mg/kg

77.4 ( 11.2 16.9 ( 3.45 13.8 ( 2.42 14.5 ( 1.57 64.2 ( 10.3 155 ( 45.0

50 mg/kg

male SD, ip

173 ( 9.10 32.3 ( 5.27 30.3 ( 3.37 26.9 ( 1.73 153 ( 35.0 377 ( 94.5

100 mg/kg

Table 3. Levels of Individual HQ Protein-S Adducts (picomoles per milligram of protein) in Kidneys from Groups of Four Male or Female F-344 Rats or Male SD Rats Treated Orally (single administration) with HQ at 0, 25, 50, and 100 mg/kg and from Groups of Four Male SD Rats Treated ip at Equivalent Dose Levelsa

a

mono 10.9 ( 3.45 2,3-di 2.96 ( 2.13 2,5-di 1.92 ( 2.52 2,6-di 1.17 ( 0.503 tri 0.910 ( 0.213 tetra 2.06 ( 0.583

adduct

Table 2. Levels of Individual HQ Protein-S Adducts (picomoles per milligram of protein) in Livers from Groups of Four Male or Female F-344 Rats or Male SD Rats Treated Orally (single administration) with HQ at 0, 25, 50, and 100 mg/kg and from Groups of Four Male SD Rats Treated ip at Equivalent Dose Levelsa

HQ-Protein Adducts following Administration to Rats Chem. Res. Toxicol., Vol. 13, No. 9, 2000 865


17.6 ( 8.22 144 ( 258 1.66 ( 1.29 29.1 ( 49.1 ND 11.8 ( 20.2

50 mg/kg

male F-344, oral

25 mg/kg

9.16 ( 4.22 5.72 ( 1.86 19.3 ( 6.47 24.0 ( 14.7 19.6 ( 7.00 19.1 ( 19.3 3.68 ( 1.28 3.90 ( 2.49 ND ND 2.32 ( 2.69 0.522 ( 1.04

0 mg/kg

0 mg/kg

28.9 ( 7.56 5.31 ( 6.15 18.6 ( 6.18 ND 8.09 ( 2.90 ND 9.01 ( 3.38 0.720 ( 0.875 2.76 ( 1.06 ND 4.94 ( 1.82 1.92 ( 2.26

100 mg/kg 31.1 ( 33.0 ND ND 3.54 ( 5.59 1.98 ( 3.97 2.61 ( 5.22

29.6 ( 7.55 ND 4.13 ( 4.84 6.62 ( 1.53 3.38 ( 0.598 ND

50 mg/kg

female F-344, oral 25 mg/kg 68.8 ( 15.2 2.24 ( 3.88 12.9 ( 11.1 16.4 ( 1.31 6.14 ( 5.32 12.8 ( 11.1 ND ND ND ND ND ND

100 mg/kg 0 mg/kg

100 mg/kg

0 mg/kg

3.69 ( 3.26 14.4 ( 5.91 18.7 ( 6.29 5.53 ( 1.92 ND ND ND ND ND ND ND ND 0.866 ( 1.00 4.18 ( 0.842 4.99 ( 0.797 ND ND 0.340 ( 0.680 0.461 ( 0.922 ND ND ND ND ND

50 mg/kg


34.8 ( 3.03 87.0 ( 5.77 ND 4.34 ( 0.290 5.40 ( 0.288 13.1 ( 0.942 7.94 ( 0.840 14.9 ( 1.34 3.21 ( 0.621 6.173 ( 0.582 2.32 ( 4.64 14.3 ( 1.11

50 mg/kg

male SD, ip 25 mg/kg

185 ( 14.2 10.6 ( 1.05 30.6 ( 7.61 33.6 ( 2.61 14.3 ( 1.51 22.1 ( 1.84

100 mg/kg

a

50 mg/kg

male F-344, oral

25 mg/kg

2.94 ( 0.157 3.06 ( 2.16 6.41 ( 2.23 ND ND ND ND ND ND ND ND 1.27 ( 1.47 ND ND 0.435 ( 0.869 ND ND ND

0 mg/kg



adduct

0 mg/kg 14.9 ( 5.17 2.18 ( 2.53 5.39 ( 1.86 5.55 ( 1.47 4.71 ( 1.21 ND

50 mg/kg


16.7 ( 4.39 3.81 ( 2.11 5.72 ( 2.21 ND ND ND 4.80 ( 1.35 ND 1.11 ( 1.32 5.00 ( 1.06 ND 2.43 ( 0.668 3.76 ( 0.844 ND 1.76 ( 1.18 ND ND 0.825 ( 1.65

100 mg/kg

0 mg/kg

10.2 ( 3.13 ND 1.03 ( 2.05 2.59 ( 0.737 1.22 ( 0.884 ND

50 mg/kg


27.2 ( 7.78 4.23 ( 1.42 4.88 ( 1.20 6.95 ( 1.51 ND ND 8.79 ( 2.86 ND ND 7.55 ( 5.24 ND ND 7.87 ( 2.53 ND ND 3.58 ( 4.18 ND ND

100 mg/kg

0 mg/kg 26.0 ( 15.0 3.81 ( 0.895 3.14 ( 4.11 ND 6.70 ( 6.23 ND 6.94 ( 4.35 ND 4.36 ( 2.64 ND ND 0.838 ( 1.68

100 mg/kg

28.7 ( 4.91 3.75 ( 2.87 7.40 ( 0.944 6.19 ( 0.862 3.87 ( 0.355 ND

25 mg/kg

65.0 ( 12.9 7.28 ( 1.78 16.1 ( 3.53 10.7 ( 3.01 7.85 ( 1.77 5.44 ( 0.865

50 mg/kg

male SD, ip

208 ( 54.7 21.9 ( 7.55 52.7 ( 19.0 29.4 ( 10.3 24.5 ( 8.33 11.9 ( 3.03

100 mg/kg

Table 5. Levels of Individual HQ Protein-S Adducts (picomoles per milligram of protein) in Blood from Groups of Four Male or Female F-344 Rats or Male SD Rats Treated Orally (single administration) with HQ at 0, 25, 50, and 100 mg/kg and from Groups of Four Male SD Rats Treated ip at Equivalent Dose Levelsa

a


adduct

Table 4. Levels of Individual HQ Protein-S Adducts (picomoles per milligram of protein) in Spleens from Groups of Four Male or Female F-344 Rats or Male SD Rats Treated Orally (single administration) with HQ at 0, 25, 50, and 100 mg/kg and from Groups of Four Male SD Rats Treated ip at Equivalent Dose Levelsa

866 Chem. Res. Toxicol., Vol. 13, No. 9, 2000 Boatman et al.


Chem. Res. Toxicol., Vol. 13, No. 9, 2000 867

Table 6. Levels of Total Protein-S Adducts (picomoles per milligram of protein) in Blood, Kidneys, Livers, and Spleens from Groups of Five Male or Female F-344 or Male SD Rats Treated Orally for a 6 Week Period (5 days/week) with HQ at 0 (control), 25, or 50 mg/kg total protein adduct level (pmol/mg of protein) ((1 SD) male F-344, oral 0 mg/kg 25 mg/kg 50 mg/kg female F-344, oral 0 mg/kg 25 mg/kg 50 mg/kg male SD, oral 0 mg/kg 25 mg/kg 50 mg/kg

blood

kidney

liver

spleen

10.3 ( 3.77 53.0 ( 28.7a 130 ( 18.4a

9.18 ( 0.630 112 ( 13.3a,c 170 ( 23.3a,c

21.4 ( 1.50 309 ( 58.7a 472 ( 177a

56.1 ( 28.1 85.1 ( 14.3a,c 81.7 ( 17.0a

10.7 ( 2.46 129 ( 23.1a,b 230 ( 51.0a,b

18.0 ( 4.48 139 ( 30.3a 308 ( 47.8a,b

74.0 ( 1.49a 566 ( 80.0a,b 904 ( 62.5a,b

66.8 ( 7.26 102 ( 17.3a,d 174 ( 29.2a,b

11.8 ( 2.32 60.7 ( 16.8a 128 ( 18.0a

9.07 ( 5.99 72.1 ( 16.2a 140 ( 29.7a

13.7 ( 7.68 261 ( 128a 449 ( 94.5a

11.9 ( 10.4e 29.7 ( 14.1 96.2 ( 18.6a

a Value statistically significantly different from the control value (p e 0.05). b Values for females were statistically significantly different from those for males at this dose level (p e 0.05). c The value for male F-344 rats was statistically significantly different from that for male SD rats at this dose level (p e 0.05). d The value for female F-344 rats was statistically significantly different from that for male SD rats at this dose level (p e 0.05). e The value for male SD rats (control) was statistically significantly different from that for male or female F-344 rats (p e 0.05).

Table 7. Levels of Individual HQ Protein-S Adducts (picomoles per milligram of protein) in Livers from Groups of Five Male or Female F-344 Rats or Male SD Rats Treated Orally for 6 Weeks (5 day/weeks) with HQ at 0, 25, and 50 mg/kga male F-344

female F-344

male SD

adduct

0 mg/kg

25 mg/kg

50 mg/kg

0 mg/kg

25 mg/kg

50 mg/kg

0 mg/kg

25 mg/kg

50 mg/kg


11.5 ( 0.746 2.81 ( 0.784 2.22 ( 1.13 1.15 ( 0.521 0.946 ( 0.217 2.78 ( 0.386

236 ( 50.6 10.6 ( 2.17 6.95 ( 6.23 10.3 ( 3.68 12.6 ( 2.13 31.8 ( 7.11

367 ( 127 14.0 ( 3.72 18.2 ( 16.5 15.4 ( 13.1 17.9 ( 9.76 39.1 ( 20.4

47.0 ( 1.78 4.94 ( 0.965 3.59 ( 0.787 5.80 ( 0.475 3.69 ( 0.198 8.92 ( 0.927

425 ( 58.5 18.2 ( 2.71 11.7 ( 12.5 36.6 ( 10.6 26.6 ( 4.68 48.3 ( 8.11

681 ( 33.8 25.1 ( 1.30 44.3 ( 20.2 40.3 ( 25.1 41.6 ( 1.01 71.7 ( 4.15

8.30 ( 4.75 0.602 ( 0.828 0.973 ( 0.625 1.50 ( 0.591 0.659 ( 0.472 1.68 ( 1.59

188 ( 91.9 6.41 ( 2.83 17.3 ( 8.69 16.7 ( 12.1 11.2 ( 4.70 21.4 ( 8.87

342 ( 66.7 10.1 ( 2.28 26.2 ( 6.91 25.3 ( 12.6 16.3 ( 3.34 29.5 ( 5.11

a


Table 8. Levels of Individual HQ Protein-S Adducts (picomoles per milligram of protein) in Kidneys from Groups of Five Male or Female F-344 Rats or Male SD Rats Treated Orally for 6 Weeks (5 days/week) with HQ at 0, 25, and 50 mg/kga male F-344

female F-344

adduct

0 mg/kg

25 mg/kg

50 mg/kg

0 mg/kg

25 mg/kg


1.61 ( 0.930 1.87 ( 0.184 0.456 ( 0.625 ND 1.33 ( 0.175 3.92 ( 0.36

13.0 ( 3.60 7.33 ( 1.64 3.90 ( 0.856 6.69 ( 1.38 23.0 ( 2.20 58.6 ( 8.80

21.5 ( 3.36 11.4 ( 2.72 7.07 ( 0.475 9.24 ( 1.35 37.1 ( 3.95 83.3 ( 14.9

2.64 ( 0.295 3.43 ( 1.95 1.38 ( 2.07 1.67 ( 0.195 2.73 ( 0.204 6.09 ( 0.32

26.9 ( 2.60 10.2 ( 1.98 4.81 ( 0.748 7.15 ( 1.38 24.3 ( 3.12 66.0 ( 25.2

a

male SD 50 mg/kg

0 mg/kg

70.3 ( 18.6 2.80 ( 2.96 21.9 ( 3.79 0.689 ( 0.954 12.2 ( 2.78 ND 17.7 ( 2.09 ND 56.1 ( 6.51 1.17 ( 0.760 129 ( 17.2 4.41 ( 2.74

25 mg/kg

50 mg/kg

11.7 ( 1.58 4.54 ( 0.965 2.16 ( 1.08 3.28 ( 1.80 15.2 ( 3.42 35.3 ( 8.94

27.9 ( 10.9 8.98 ( 2.14 7.07 ( 2.00 5.41 ( 2.14 29.8 ( 8.21 60.7 ( 13.8


the highest levels of total protein-S adducts; values for total adducts in spleens from female rats ranged from 66.8 (untreated) to 174 pmol/mg (50 mg/kg). Comparable values for spleen adducts in male F-344 rats ranged from 56.1 (untreated) to 81.7 pmol/mg (50 mg/kg). Assays of spleen proteins from male and female F-344 rats were compromised by high levels of interfering substances chromatographing in the region containing the di-adducts, resulting in inflated levels of these adducts at all dose levels, including the controls. Male SD rats had generally lower levels of total HQ protein-S adducts in spleens with values ranging from 11.9 (untreated) to 96.2 pmol/mg (50 mg/kg). Unlike male F-344 rats, a clear dose-response relationship was seen for the amount of total protein-S adducts in spleens of male SD rats. Although the level of total HQ adducts was significantly increased as a result of oral administration for 6 weeks, the relative proportions of individual HQ adducts (mono, di, tri, and tetra) were similar (with the exception of blood) to those observed following a single oral administration and varied as a function of tissue. In liver,

the mono-adduct was the predominant form and accounted for >72% of the protein-S-bound HQ in this tissue (Table 7). In the kidneys, the sum of the tri- and tetra-adducts accounted for >60% of the total in this tissue (Table 8). In spleens (male F-344 excluded), the mono-adduct again predominated (Table 9). For a given tissue (with the exception of the results for spleens from male F-344 rats), the relative proportions of the various adducts did not vary as a function of either gender or strain of rat. In the case of blood protein, total HQ adduct levels in the 6 week study were increased from 7- to 18-fold over comparable levels from the single-dose study (compare Tables 1 and 6). In addition, single oral administrations produced primarily mono-protein-S adducts with relatively smaller amounts of the di- and tri-adducts. However, following oral treatment for 6 weeks, larger amounts of di-, tri-, and tetra-adducts were formed (Table 10). The tetra-adduct was also increased in F-344 rats compared to SD rats, reaching levels comparable to that of the mono-adduct.

868


Boatman et al.

Table 9. Levels of Individual HQ Protein-S Adducts (picomoles per milligram of protein) in Spleens from Groups of Five Male or Female F-344 Rats or Male SD Rats Treated Orally for 6 Weeks (5 days/week) with HQ at 0, 25, and 50 mg/kga male F-344

female F-344

male SD

adduct

0 mg/kg

25 mg/kg

50 mg/kg

0 mg/kg

25 mg/kg

50 mg/kg

0 mg/kg

25 mg/kg

50 mg/kg


3.04 ( 2.18 20.7 ( 10.7 19.5 ( 10.6 3.91 ( 1.56 ND 8.89 ( 6.44

13.3 ( 2.48 26.5 ( 4.63 23.4 ( 5.05 6.64 ( 2.01 5.30 ( 1.14 9.91 ( 1.81

22.6 ( 3.67 19.7 ( 5.17 11.9 ( 6.57 7.69 ( 1.96 8.22 ( 1.75 11.5 ( 7.02

7.92 ( 1.60 21.1 ( 7.08 20.1 ( 9.98 10.9 ( 12.5 ND 6.71 ( 6.17

26.2 ( 6.69 22.8 ( 6.76 16.6 ( 9.66 10.7 ( 1.41 9.35 ( 1.46 16.6 ( 3.08

50.9 ( 8.25 25.7 ( 8.73 17.6 ( 7.56 22.2 ( 3.50 21.3 ( 3.08 36.3 ( 6.92

5.44 ( 3.75 ND 2.24 ( 2.11 ND ND 4.22 ( 5.78

14.7 ( 5.86 2.18 ( 3.27 2.18 ( 3.27 5.14 ( 1.54 3.31 ( 0.718 2.23 ( 4.98

35.5 ( 8.03 9.88 ( 3.78 15.5 ( 4.04 12.5 ( 3.35 8.16 ( 1.93 14.7 ( 3.07

a


Table 10. Levels of Individual HQ Protein-S Adducts (picomoles per milligram of protein) in Blood from Groups of Five Male or Female F-344 Rats or Male SD Rats Treated Orally for 6 Weeks (5 days/week) with HQ at 0, 25, and 50 mg/kga male F-344 adduct mono 2,3-di 2,5-di 2,6-di tri tetra a

0 mg/kg

25 mg/kg

female F-344 50 mg/kg

0 mg/kg

25 mg/kg

4.50 ( 1.79 12.15 ( 6.74 38.5 ( 4.11 2.84 ( 0.215 34.9 ( 6.94 4.57 ( 1.44 7.77 ( 4.68 18.9 ( 2.30 2.93 ( 0.76 21.9 ( 3.81 ND 6.25 ( 4.63 14.0 ( 2.70 0.880 ( 0.812 13.6 ( 2.68 0.759 ( 0.711 3.84 ( 2.43 10.4 ( 1.59 1.12 ( 0.059 12.5 ( 2.13 ND 7.13 ( 4.79 17.7 ( 3.35 1.22 ( 0.135 18.8 ( 3.81 0.446 ( 0.998 15.8 ( 8.10 30.6 ( 5.20 1.66 ( 1.54 27.8 ( 5.67

male SD 50 mg/kg

0 mg/kg

65.6 ( 15.3 2.90 ( 0.802 31.9 ( 6.52 3.79 ( 1.02 24.9 ( 5.03 1.96 ( 0.865 20.0 ( 4.99 0.706 ( 0.070 35.1 ( 7.11 0.318 ( 0.297 52.2 ( 13.6 2.16 ( 0.599

25 mg/kg

50 mg/kg

20.7 ( 6.54 9.64 ( 2.76 7.29 ( 2.61 5.29 ( 0.978 7.56 ( 1.90 10.2 ( 2.39

48.8 ( 8.30 18.8 ( 3.28 16.3 ( 1.48 10.6 ( 2.96 14.9 ( 1.85 18.8 ( 1.95


Table 11. Levels of the 2,5-Di-, 2,6-Di-, and Tri-Protein-S Adducts (picomoles per milligram of protein) in Protein Isolated from the Urine of Male F-344 and SD Rats Treated Orally for 6 Weeks (5 days/week) with HQ at 25 or 50 mg/kg dose level (mg/kg) male F-344 25a 50c male SD 25d 50a

protein adduct level (pmol/mg of protein) 2,5-di 2,6-di tri sum NDb 116 ND ND

177 208

269 138

446 463

83.7 170

153 52.0

237 222

a Values represent the mean results from two animals. b N. D. ) none detected. c Values represent the mean results from three animals. d Values obtained for a single animal.

Small amounts of urinary proteins were isolated from male F-344 and SD rats treated at 25 or 50 mg/kg levels (Table 11). The amounts of protein isolated from female F-344 rats were negligible. Sufficient quantities of protein were obtained from certain animals to allow analysis by permethylation. Interestingly, only di- and tri-adducts were detected in these samples. Male F-344 rats had the highest concentrations of protein-S adducts, reaching mean levels of 446 (25 mg/kg, results for two animals) and 463 pmol/mg (50 mg/kg, results for three animals). Male SD rats attained mean levels of 237 (25 mg/kg, results for a single animal) and 222 pmol/mg (50 mg/kg, results for two animals). (3) Urinary End Points. Summarized in Table 12 are urine volumes and levels of urinary glucose, creatinine, and marker enzymes for rats treated orally with HQ for 6 weeks. No dose-dependent variations in any of the measured end points were noted for either urine volume or urinary levels of glucose, creatinine, AAP, NAG, or γ-GT. Similarly, results for urinary levels of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1R (PGF1) indicate no dose-related changes (Table 13). Female F-344 rats had statistically significantly higher levels of both TXB2 and PGF1 than did male rats. However, when the data were expressed as a ratio of TXB2 to PGF1, no significant

dose, strain, or gender-related differences were detected (Table 14).

Discussion HQ is nephrotoxic in F-344 rats following oral administration, resulting in renal tubular cell degeneration and mild enzymuria and glucosuria (1, 2). Additionally, the female F-344 rat is more sensitive to the acute nephrotoxicity of HQ than is the male, whereas SD rats are insensitive (1). In contrast, the severity of nephrotoxicity is greater in males than females following repeated treatments (2, 3). The cytotoxicity of certain quinones has been attributed to their ability to either produce oxidative stress, as a result of redox cycling, or by the arylation of critical cellular macromolecules (23). It has recently been recognized that certain quinones produce cytoxicity exclusively by one of these mechanisms (24). In the case of 2,3-dimethoxynaphthoquinone, for example, cytotoxicity has been correlated exclusively with redox cycling (25). In contrast, BQ, which is formed through the oxidation of HQ, does not redox cycle in isolated mitochondria, microsomes, or isolated hepatocytes and is cytotoxic through its arylating activity (24). A minimal role for redox cycling in HQ renal toxicity is suggested by recent work indicating only a slight depression of glutathione and cysteine levels in the kidneys of male F-344 rats given near lethal doses of HQ as well as the lack of an increase in the level of oxidized glutathione in these rats (26). In addition, no increases in 8-hydroxydeoxyguanosine levels have been detected in DNA extracted from the kidneys of male F-344 rats given HQ (27). Recently, it has been proposed that the increased incidence and severity of chronic progressive nephropathy (CPN) in the male F-344 rat may lead to the induction of renal adenomas in older male rats of this strain (5). 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.



Table 12. Urine Volumes and Urinary Levels of Glucose, Creatinine, and Marker Enzymes from Groups of Five Male or Female F-344 or Male SD Rats at the End of a 6 Week Oral Treatment with HQ at 0, 25, or 50 mg/kga rat strain

dose

volume (mL/24 h)

AAP (munit/h)

NAG (munit/h)

γ-GT (munit/h)

glucose (mg/h)

creatinine (mg/h)

0 25 50 0 25 50 0 25 50

10.00 ( 0.86 8.60 ( 2.68 10.06 ( 0.49 10.74 ( 3.88 10.28 ( 2.69 10.24 ( 3.21 21.82 ( 4.15 19.9 ( 7.69 20.26 ( 7.93

9.21 ( 1.12 8.36 ( 0.75 9.05 ( 0.55 2.86 ( 0.35 2.57 ( 0.39 2.56 ( 0.20 10.79 ( 1.87 9.85 ( 2.02 11.40 ( 2.81

6.43 ( 0.37 6.14 ( 0.82 6.43 ( 0.48 4.27 ( 0.17 4.45 ( 0.24 4.99 ( 0.26 15.56 ( 2.02 18.04 ( 3.71 17.01 ( 2.75

654 ( 97 606 ( 36 589 ( 45 298 ( 35 205 ( 61 205 ( 17 1266 ( 423 1537 ( 520 1326 ( 277

0.19 ( 0.05 0.16 ( 0.02 0.17 ( 0.03 0.09 ( 0.01 0.09 ( 0.01 0.09 ( 0.01 0.24 ( 0.08 0.41 ( 0.11 0.35 ( 0.09

0.43 ( 0.05 0.41 ( 0.04 0.40 ( 0.03 0.29 ( 0.02 0.29 ( 0.03 0.29 ( 0.02 0.78 ( 0.08 0.70 ( 0.04 0.68 ( 0.05

male F-344 female F-344 male SD

a


Table 13. Urine Levels of Thromboxane B2 (TXB2) and 6-Keto-Prostaglandin F1r (PGF1) from Groups of Five Male and Female F-344 and Male SD Rats at the End of a 6 Week Oral Treatment with HQ at 0, 25, or 50 mg/kga

rat strain male F-344 female F-344 male SD

dose 0 25 50 0 25 50 0 25 50

amount produced (ng/d/kg of body weight) TXB2 PGF1 75.9 ( 10.2 61.6 ( 8.5 69.4 ( 11.1 127.0 ( 35.6b 112.2 ( 20.0b 115.8 ( 13.4b 57.1 ( 24.2 51.6 ( 12.8 59.0 ( 8.5

121.2 ( 17.3 111.7 ( 21.2 109.4 ( 16.4 286.2 ( 67.1b 231.7 ( 24.2b 219.2 ( 18.0b 111.1 ( 15.9 82.8 ( 15.3 106.9 ( 14.9

TXB2/PGF1 ratio 0.631 ( 0.075 0.579 ( 0.164 0.634 ( 0.048 0.473 ( 0.168 0.499 ( 0.147 0.530 ( 0.055 0.519 ( 0.212 0.635 ( 0.172 0.552 ( 0.033

a The error represents (1 SD. b Female F-344 rats had statistically significanlty higher levels of both TXB2 and PGF1 at all dose levels than did either male F-344 or male SD rats (p e 0.05).

Table 14. Comparison of Thromboxane B2 (TXB2) and 6-Keto-Prostaglandin F1r (PGF1) Levels with Levels of Total HQ Protein-S Adducts in Kidney Protein from Groups of Five Male and Female F-344 and Male SD Rats at the End of a 6 Week Oral Treatment with HQ at 50 mg/kg amount produced HQ protein-S (ng/d/kg of body weight) adduct (pmol/mg of protein) TXB2a PGF1a male F-344 (ratio)b 69.0 (1.23) 113.8 (1.13) female F-344 (ratio) 118.3 (2.12) 245.7 (2.45) male SD (ratio) 55.9 (1.00) 100.3 (1.00)

170 (1.21) 308 (2.20) 140 (1.00)

a Mean value for 12 or 13 animals. b Ratio of the value to that found in male SD rats (normalized to 1.00).

Background levels of HQ protein-S adducts in blood, kidneys, and livers from rats ranged from 3 to 74 pmol/ mg of protein (Tables 1 and 6) with the highest levels found in livers from female F-344 rats. These values compare favorably to background levels of HQ protein-S adducts of 12 and 26 pmol/mg of protein reported by McDonald et al. (28) for rat hemoglobin and bone-marrow proteins, respectively. Such results suggest a significant exposure to HQ, BQ, or a metabolic precursor of these as a result of diet or due to endogenous production. In this regard, recent work from this laboratory has shown that significant human exposure to HQ results from plant-derived dietary sources, including items such as coffee, wheat-based products, pears, and red wine (29). As summarized in Tables 1-5, the levels of total protein-S adducts of HQ were highest in livers following single oral administrations of HQ. As evidenced from this study, rats are capable of maintaining significant levels of protein-bound HQ in liver without the concomitant production of hepatotoxicity. In the current studies, levels of protein-bound HQ in livers from male and female

F-344 rats were 478 and 1003 pmol/mg of protein, respectively, following administration of a single, nonhepatotoxic oral dose of 100 mg/kg (Table 1). As a comparison, protein-bound levels of approximately 400 pmol/mg of protein in livers from male F-344 rats were found for acetaminophen following a single subcutaneous administration of 500 mg/kg, a dose which produces hepatic necrosis (30). The relative proportions of the individual HQ adducts (i.e., mono, di, tri, or tetra) were found not to vary with dose but to be strongly dependent on tissue type. A striking difference in the pattern of adducts was seen between liver and kidney. Thus, in male F-344 rats receiving a single 100 mg/kg oral dose, the mono-adduct accounted for 84% of the total adducts found in livers at this dose level (Table 2) and suggests that the liver is primarily responsible for the conversion of parent HQ to metabolites derived from oxidation to BQ. Given the significant, albeit lower, proportion of protein monoadduct seen in the kidney, it is reasonable to suggest that the resultant distribution of more highly conjugated adducts in this tissue results from further oxidative conversion and conjugation within this organ (tri- and tetra-substituted adducts accounting for 72% of the total kidney adducts; Table 3). Alternatively, extrarenal conversion of glutathionyl adducts, initially formed in the liver and possibly in the blood (31), may undergo further bioconversion within the kidney and as a consequence lead to elevated levels of the more highly substituted HQ adducts seen in this tissue. In the case of blood protein, primarily mono-adducts with protein are detected following a single 100 mg/kg oral dose (Table 5) with only slight additional amounts of the di- and trisubstituted adducts detected. Single ip administration of HQ in male SD rats produced significantly higher levels of total protein-S adducts compared to oral administration in most tissues examined but did not change the relative proportions of the individual adducts (see Tables 2-5). At a dose of 100 mg/kg, total adduct levels in blood, kidneys, and spleens following ip administration to male SD rats were 5-10fold higher than for comparable oral doses (see Table 1). In contrast, adduct levels in livers following ip dosing were only 0.9-1.9 times those of comparable oral values, suggesting that extrahepatic activation may account for the majority of the protein binding seen in kidneys, blood, and spleens. In addition, ip administration results in the bypassing of the detoxification pathways of sulfation and glucuronidation, which are present in the gut (32), leading to a greater extent of formation of glutathionyl adducts. As was the case following single gavage or ip administrations of HQ, the relative proportions of the individual

870


HQ protein-S adducts in livers and kidneys following repeated gavage treatment strongly depended on tissue type (see Tables 7 and 8). For spleens and blood (Tables 9 and 10), repeated administration resulted in increased levels of the more highly conjugated protein-S adducts versus that observed following a single HQ administration. The mono-adduct accounts for the majority of the total adducts found in livers from multiply treated rats (Table 7) and presumably results from the conversion of HQ to BQ and subsequent protein binding. As was also the case following single administration of HQ, increased levels of tri- and tetrasubstituted protein-S adducts were found in kidneys. In the case of blood protein, repetitive treatment leads to greater increases in the levels of total HQ adducts than in other tissues and also to a greater proportion of the more highly conjugated adducts than was observed following single gavage administrations (compare values in Tables 5 and 10). Longer relative half-lives of the HQ adducts in blood versus other tissues provide one explanation for increased total adduct levels. Additionally, enterohepatic cycling of glutathionyl conjugates of HQ provides a possible explanation for both the increased adduct levels and the increased proportions of the more highly conjugated adducts. With regard to the latter possibility, Hill et al. (33) have shown that mono-, di-, and tri-glutathione conjugates of HQ in bile account for the majority (67%) of total HQ-sulfhydryl conjugates quantitated in urine and bile following a single ip dose. Protein-S adduct levels in spleens generally reflected the levels found in blood (Table 9). Male F-344 rats treated orally for 6 weeks clearly exhibited increasing levels of mono- and tri-adducts in this organ. The general correspondence of adduct levels in spleens with those in blood may reflect the spleen’s function as a storage area for red blood cells (34). The nephrotoxicity of HQ-glutathione conjugates increases with increased substitution (up to three substituents) when administered iv to SD rats (12). Thus, levels of tetrasubstituted adducts to protein may serve as a tissue dosimeter for these more highly substituted and potentially more toxic metabolites. Plotted in Figures 1 and 2 are tetrasubstituted adduct levels in livers, blood, and kidneys following a single administration of HQ (50 mg/kg, oral and ip) or following oral administration for 6 weeks at 50 mg/kg. These results are representative of effects seen at all dose levels. Higher levels of tetrasubstituted adducts were seen in liver and kidney proteins from female F-344 rats than in other orally treated groups following either single or repeated administration. Ip administration in male SD rats yielded levels of tetrasubstituted adducts to kidney protein approximately 2-3-fold higher than in animals treated orally but yielded levels in livers roughly comparable to those in other groups (Figure 1). Detectable levels of tetrasubstituted protein-S adducts in blood were only seen following ip administration (Figure 1). However, following a 6 week administration at this same dose, tetrasubstituted adduct levels in blood nearly equaled those in liver (Figure 2) with female F-344 rats again showing the highest levels. Tetrasubstituted adduct levels for male F-344 rats were consistently greater than those of male SD rats in the 6 week studies for all three tissues (Figure 2). Inhibition of mitochondrial function has been proposed to explain the nephrotoxicity of glutathione conjugates

Boatman et al.

of haloalkenes and quinones (35, 36). Inactivation or alkylation of protein sulfhydryl groups may play a role in this loss of function. Thus, the potent nephrotoxicant derived from HQ, 2,3,5-(triglutathion-S-yl)hydroquinone, inhibits renal mitochondrial function either in vitro or in vivo by a mechanism which appears to involve the unmetabolized conjugate and may result from arylation of critical mitochondrial proteins (36). In vivo, the effects observed with 2,3,5-(triglutathion-S-yl)hydroquinone include an early elevation of state 4 respiration followed by a later depression of state 3 respiration and a corresponding loss of mitochondrial activity (36). Arylation of rat liver mitochondrial protein sulfhydryls by BQ in vitro has also been associated with decreased state 3 respiration (37). Sulfhydryl-dependent enzymes are inhibited in rat hepatic mitochondria by reaction with BQ with a 40% depletion (caused by 40 µM BQ), resulting in mitochondrial Ca2+ release and inhibitions of the mitochondrial enzymes phosphate translocase and isocitrate dehydrogenase but not of the cytosolic enzyme succinate dehydrogenase (37). In this regard, sulfhydryl group modification of mitochondrial proteins has also been proposed as a mechanism for adriamycin cardiotoxicity (38). Several urinary end points of nephrotoxicity were measured in the current studies. Prolonged gavage treatment with HQ produced no diuresis or any significant changes in the rates of excretion of glucose, creatinine, AAP, NAG, or γ-GT (Table 12). This is consistent with previous work (3) from this laboratory in which a 6 week treatment regimen administered to similarly aged animals at equivalent doses produced only slight elevations in urinary enzyme levels in male rats (at 6 weeks only). In small amounts of precipitable protein isolated from urine from male rats in the current studies, only di- and tri-adducts were found (Table 11). Recorded levels of total adducts in urine from male F-344 rats were higher than those for male SD rats; however, data from too few animals were obtained to ascertain the statistical significance of this finding. Female rats lack appreciable urinary protein and also display a more pronounced acute kidney response to HQ. In male rats, arylation by HQ metabolites of protein destined for excretion may protect male kidneys from acute HQ toxicity. Whether such covalently modified proteins contribute to chronic insult in the male F-344 rat is unknown. In the current report, urinary levels of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1R (PGF1) were measured for rats treated for 6 weeks with HQ at levels up to 50 mg/kg/day (Table 13). No significant dose-related changes in these marker compounds were detected in any of the treatment groups. To the extent that these materials result from renal synthesis, it can be surmised that renal hemodynamics were unaltered by this HQ treatment (39). However, female F-344 rats were found to exhibit levels of TXB2 and PGF1 approximately double the levels found in either male F-344 or SD rats. Interestingly, this increased production of TXB2 and PGF1 correlates directly with increased protein-S adduct levels in kidneys, which are more than doubled for female F-344 rats versus levels in male rats (Table 14). Also, male F-344 rats exhibit corresponding but small increases over male SD rats in these same end points. Such findings suggest that female F-344 rats (and possibly male F-344 rats) have greater levels of the prostaglandin H synthase (PHS) enzyme complex which, through its peroxidase activity, has been shown to oxidize HQ (40)


and several other nephrotoxicants, including 2-bromohydroquinone (41) and acetaminophen (42). Also consistent with a role for PHS activity in the renal bioactivation of HQ is the observation that older (10 months of age) rats are less sensitive to HQ nephrotoxicity than young adult rats (6), since a progressive decrease in rat renal PHS activity with age has been reported (43). Oxidation of HQ or HQ thioether conjugates by PHS to reactive intermediates within the kidney could provide a mechanism for the acute toxicity observed as well as explain the elevated levels of protein-S adducts seen in female F-344 rats.

Conclusions Elevated protein-S adduct levels in kidneys from female F-344 rats versus levels in male F-344 or SD rats are consistent with the differences in the acute nephrotoxicity of HQ seen in these strains and genders of rats (1). Specifically, the order of sensitivity is as follows: female F-344 > male F-344 > male SD. In addition, increased levels of the tri- and tetrasubstituted protein-S adducts found in kidneys are consistent with the increased nephrotoxicity of the di- and tri-glutathione conjugates of HQ, which may represent the proximate renal toxicants. In addition, conversion of HQ or HQ conjugates within the kidney may explain the increased sensitivity of this organ. Although significantly elevated HQ protein-S adduct levels were detected in protein from kidneys, this did not correlate with measurable biochemical indices of nephrotoxicity. Following single or repeated gavage administration of HQ, female F-344 rats had higher levels of HQ protein-S adducts in blood, livers, and kidneys than did male rats. Male F-344 rats had elevated adduct levels in these same tissues as compared to male SD rats. Moreover, the SD rat, which is susceptible to nephrotoxicity when treated ip, had significantly higher total adduct levels after ip treatment than after oral treatment. Extrahepatic conversion of HQ or HQ conjugates may account for the 5-10-fold higher levels of protein-S adducts in blood, kidneys, and spleens following ip administration versus corresponding increases of only 1-2-fold in livers. Results from the study presented here provide further evidence for a role of the prostaglandin H synthase complex as a possible catalyst in the metabolic activation of HQ. Protein arylation by HQ or HQ metabolites alone cannot account for the greater sensitivity of the male F-344 rat toward the chronic toxicity of HQ. However, susceptibility to nephrotoxic effects is certain to be influenced by sex differences in the progression of age-related rat nephropathy. In this regard, the arylation of renal proteins may lead to an enhancement of CPN in aged male F-344 rats and may play an important role in the formation of male F-344 rat renal adenomas. Other male rat factors that may contribute to kidney tumor development include possible chronic insult due to glomerular filtration and processing of abnormal (HQ-modified) low-molecular weight proteins by the kidney, and a more permissive environment for tumor expression resulting from development of CPN. Rat strain differences in HQ metabolism consistent with an absence of long-term effects in the SD rat are currently under investigation. In the current studies, levels of the tetrasubstituted adducts provided a useful and informative tissue dosimeter for HQ.


Acknowledgment. Financial support was provided by Eastman Chemical Co., Mitsui Petrochemicals (America), Ltd., and Rhodia, Inc.

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