Chem. Res. Toxicol. 1998, 11, 1291-1300
1291
Immunochemical Analysis of Quinol-Thioether-Derived Covalent Protein Adducts in Rodent Species Sensitive and Resistant to Quinol-Thioether-Mediated Nephrotoxicity Heather E. Kleiner,†,‡ Thomas W. Jones,§ Terrence J. Monks,† and Serrine S. Lau*,† Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, and Department of Biochemical Toxicology, Eli Lilly and Company, 2001 West Main Street, Greenfield, Indiana 46140 Received June 10, 1998
2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ) is nephrotoxic in male Fischer 344 rats (20 µmol/kg) and albino guinea pigs (200 µmol/kg), but not BALB/c or B6C3F1 mice or Golden Syrian hamsters (200 µmol/kg). Since quinones are known to alkylate proteins, and because such macromolecular damage may play a role in cytotoxicity, we investigated the covalent binding of TGHQ to kidney (target tissue) and liver (nontarget tissue) of rodents “sensitive” or “resistant” to the nephrotoxic effects of TGHQ. Immunohistochemical staining of tissue obtained 2 h after administration of TGHQ, with rabbit anti-2-bromo-N-(acetyl-L-cystein-Syl)HQ antibodies, correlated with the subsequent region of necrosis observed 19 h after dosing in Fischer 344 rats and guinea pigs. Immunohistochemical staining was localized to the S3 segment of the renal proximal tubules, at the corticomedullary junction along the medullary rays, and in the outer stripe of the outer medulla. Immunostaining was also observed in the same region in hamsters, but subsequent necrosis did not develop. In contrast, no immunostaining was observed in mice. Moreover, immunostaining was not detected in the livers of any species. Western blot analysis revealed numerous immunoreactive renal proteins in TGHQtreated animals. The most distinctive immunostaining renal proteins were observed in Fischer 344 rats at ∼34 kDa (mitochondria), ∼35 kDa (nuclei) which comigrated with histone H1, and ∼73 kDa (urine) which comigrated with γ-glutamyl transpeptidase. These adducted proteins were not detected in other species. Qualitative differences in alkylated proteins may therefore contribute to species susceptibility to TGHQ.
Introduction Hydroquinone (HQ)1 is both nephrotoxic and nephrocarcinogenic in male Fischer 344 rats (1-3) but not in B6C3F1 mice (1, 2). Although the mechanism(s) of the renal effects of HQ is not clear, previous studies suggest that conjugation of HQ with glutathione (GSH) may be * To whom all correspondence should be addressed: Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712. Telephone: (512) 471-5190. Fax: (512) 471-5002. E-mail:
[email protected]. † The University of Texas at Austin. ‡ Present address: The University of Texas M. D. Anderson Cancer Center, Science Park Research Division, P.O. Box 389, Smithville, TX 78957. Telephone: (512) 237-9441. Fax: (512) 237-2444. E-mail:
[email protected]. § Eli Lilly and Company. 1 Abbreviations: ABC, avidin-biotin complex; 2-BrHQ, 2-bromohydroquinone; 2-BrGHQ, 2-bromo(glutathion-S-yl)hydroquinone; 2-BrHQ-NAC, 2-bromo-6-(N-acetyl-L-cystein-S-yl)hydroquinone; BrBGHQ, 2-bromo-3,5(6)-bis(glutathion-S-yl)hydroquinone; BSA, bovine serum albumin; DAB, diaminobenzidine; ECL, enhanced chemiluminescence; EDC, 1-ethyl-3-(3-diamethylaminopropyl)carbodiimide hydrochloride; ELISA, enzyme-linked immunosorbent assay; GSH, glutathione; γ-GT, γ-glutamyl transpeptidase; HQ, hydroquinone; TGHQ, 2,3,5-tris(glutathion-S-yl)hydroquinone; ic, intracardiac; KLH, keyholelimpet hemocyanin; P450, cytochrome P450; PBS, phosphate-buffered saline; PMSF, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl sulfate; TBS, tris-buffered saline; TFEC, S-(1,1,2,2-tetrafluoroethyl)L-cysteine.
a pathway of bioactivation. In support of this view, GSH conjugates of HQ are more potent nephrotoxicants in rats than HQ itself (4). TGHQ is the most potent conjugate, causing severe toxicity at a dose of 20 µmol/kg (iv) in rats (4), and significant increases in the urinary excretion of alkaline phosphatase, γ-glutamyl transpeptidase, and glutathione S-transferase at 7.5 µmol/kg (iv) (5). Moreover, HQ-GSH conjugates are excreted as biliary and urinary metabolites of HQ (1.8 mmol/kg ip) in rats in sufficient quantities to support a role for GSH conjugation in HQ-induced nephrotoxicity (6). Male Fischer 344 rats are sensitive to the nephrotoxicity of TGHQ (20 µmol/kg iv), whereas BALB/c mice, B6C3F1 mice, and Golden Syrian hamsters appear to be “resistant” to nephrotoxicity, even at doses 10 times higher than those used in rats to produce overt toxicity (7). Albino guinea pigs are susceptible to TGHQ-induced nephrotoxicity at relatively high doses (200 µmol/kg ic), although the nephrotoxicity is less severe than that observed in rats (7). Species differences in susceptibility to TGHQ therefore provide a useful model with which to examine the mechanistic role of covalent protein binding in tissue necrosis. Since the S3 segment of the renal proximal tubules is the selective target of TGHQ in rats (7), it is important
10.1021/tx9801357 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/29/1998
1292 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
to determine whether covalent binding of TGHQ to proteins occurs within the same region. An immunohistochemical approach has been used to assess the significance of protein alkylation in halothane, acetaminophen, and haloalkene-mediated toxicities (8-12). We have demonstrated previously that anti-2-BrHQ-NAC antibodies produced in our laboratory can detect in vivo covalent protein adducts of 2-BrHQ, HQ, and their corresponding GSH conjugates (13). Thus, the first goal of these studies was to employ these antibodies to determine the localization of TGHQ-derived covalent protein adducts in kidneys and livers (a nontarget organ) of rats, mice, guinea pigs, and hamsters. In addition, an assessment of the role of covalent binding in different renal subcellular fractions, within the context of a species comparison, permits further insight into potential mechanisms of the sitespecific toxicity caused by quinone-thioethers. Thus, our second goal was to identify putative critical macromolecules by comparing alkylation patterns in susceptible and resistant species.
Experimental Procedures Caution: The following chemicals are hazardous and should be handled carefully: HQ, TGHQ, diaminobenzidine (DAB), nickel chloride, and acrylamide. Chemicals. Bovine serum albumin (BSA) (fraction V, heatshock) was purchased from Boehringer-Mannheim Corp. (Indianapolis, IN). DAB was obtained from Sigma Chemical Co. (St. Louis, MO). Bovine kidney γ-GT (>99.9% pure) was produced by Calbiochem (La Jolla, CA). Biotinylated goat antirabbit IgG and an avidin-biotin complex (ABC) kit was obtained from Vector Laboratories (Burlingame, CA). Sucrose was a product of ICN Biomedicals Inc. (Cleveland, OH). TGHQ was synthesized in our laboratory as described previously (4). Americlear was supplied by Baxter Diagnostics, Inc. (Deerfield, IL). Enhanced chemiluminescent reagents (ECL) and Hyperfilm ECL were purchased from Amersham Life Science (Arlington Heights, IL). Animals. Male Fischer 344 rats (140-160 g), B6C3F1 mice (21-25 g), Golden Syrian hamsters (97-107 g), and albino guinea pigs (250-280 g) were obtained from Harlan SpragueDawley (Indianpolis, IN). The animals were allowed food and water ad libitum prior to and during the experiments. Animals were housed in an animal care facility with a 12 h light/dark cycle prior to and during experiments. Dosing of Animals and Isolation of Tissue. Animals were euthanized 2 h after dosing because previous studies showed maximum 2-Br-[14C]-HQ-derived protein adduct formation between 2 and 4 h (13). Male albino guinea pigs, Golden Syrian hamsters, and B6C3F1 mice were dosed with 200 µmol/kg (ic) TGHQ [dissolved in phosphate-buffered saline [PBS, 10 mM phosphate buffer (pH 7.4) containing 140 mM sodium chloride]]. Male Fischer 344 rats were dosed with 20 µmol/kg TGHQ (tail vein, iv). Control animals were treated with vehicle only. Individual animals were housed in stainless steel metabolism cages (except mice, which were housed two per cage), and urine was collected in light-protected tubes at 4 °C. Two hours after dosing, animals were anesthetized with 0.25 mL/100 g (ip) equithesin. The contents of the bladder were pooled with the urine that had already been collected. Renal toxicity was determined by assaying urinary excretion of γ-GT (Sigma Technical Bulletin 545). Livers and kidneys were perfused with ice-cold isolation buffer [20 mM Tris buffer (pH 7.4) containing 250 mM sucrose, 20 mM HEPES, 0.1 mM PMSF, and 1 mM EDTA] via the hepatic artery and aorta, respectively. With the remaining kidneys, papillae were dissected and discarded. The renal and hepatic tissues were homogenized 1:3 (w/v) in isolation buffer, and nuclei, mitochondria, plasma membrane, endoplasmic
Kleiner et al. reticulum, and cytosol-enriched fractions were isolated by differential centrifugation as described previously (13). Protein concentrations were determined by the method of Lowry (14) using BSA as a standard. Aliquots of tissue were stored at -80 °C for later immunochemical analysis. Immunohistochemistry. Immunohistochemical staining was conducted as follows. Tissue (liver or kidney) slices were preserved in zinc-buffered formalin [4% formaldehyde in PBS (pH 7.5)], embedded in paraffin, cut in 5 µm slices, and placed on poly-L-lysine-coated slides. Duplicate slices were placed on glass microscope slides and stained with hematoxylin and eosin for histopathological analysis. Slides for immunohistochemical analysis were heated to 65 °C, deparaffinized twice for 5 min each in Americlear, placed in 100% ethanol (two times for 5 min each), and then placed in 95% ethanol for 5 min. Endogenous peroxidases were inactivated in 0.3% hydrogen peroxide in methanol for 20 min. Samples were placed in PBS-BSA (0.1% BSA, fraction V, heat-shock, three times for 5 min each), boiled in a microwave oven for 5 min, cooled for 10 min, and placed in affinity-purified rabbit anti-2-BrHQ-NAC IgG (1:30 dilution) for 1 h. Slides were washed (three times for 5 min each) in PBSBSA and placed in biotinylated goat-anti-rabbit IgG (H and L) (1:227 dilution) for 30 min. Slides were washed (as above), placed in the ABC reagent for 30 min, washed again, and developed with the DAB reagent [0.778 mg/mL DAB, 0.389 mg/ mL NiCl2, and 0.009% hydrogen peroxide in 100 mM Tris buffer (pH 7.2)] for 90 s. Slides were counterstained in hematoxylin, mounted with a glass coverslip, and analyzed histopathologically. Western Blot Analysis. Proteins (75-250 µg/lane) were resolved by the procedure of Laemmli (15) and transferred to nitrocellulose according to Towbin et al. (16), as described previously (13). The immunoblot procedure was derived from the method described by Kenna and co-workers (17) with minor modifications (13).
Results Site-Selective Covalent Binding of TGHQ in the Kidney. Immunohistochemical analysis of kidney and liver sections from each species obtained 2 h after treatment with TGHQ (Figures 1 and 2) reveals selective anti-2-BrHQ-NAC immunostaining in the kidneys of all species, although staining in B6C3F1 mice is extremely faint and required longer development (Figure 1H). Images obtained with low magnification (9×) of kidney sections from Fischer 344 rats (Figure 1B), guinea pigs (Figure 1D), and hamsters (Figure 1F) treated with TGHQ show immunostaining localized to the S3 segment of the renal proximal tubules, at the corticomedullary junction along the medullary rays, and in the outer stripe of the outer medulla (OSOM). In contrast, immunostaining is not detectable in kidney sections from vehicle control animals (panels A, C, E, and G of Figure 1). Immunostained cells in the proximal tubules are clearly visible in kidney sections from Fischer 344 rats (Figure 2A), guinea pigs (Figure 2B), and hamsters (Figure 2C) treated with TGHQ. Intense staining of the apical (brush border) membrane is evident in the rat (Figure 2A) and to a lesser extent in the guinea pig (Figure 2B). In addition, shedding of the apical membrane into the lumen is evident in both the rat and guinea pig, and correlates with increased γ-GT activity in urine (7). Staining of the apical membrane in proximal tubules of the hamster is also apparent, although immunostaining appears to be more diffuse (Figure 2C). Immunostained lumenal debris is also apparent in hamster tubules (Figure 2C). No immunostaining is detected in either treated or untreated livers (data not shown) in any of the species examined.
Species Differences in Quinone-Thioether Adducts
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1293
Figure 1. Immunohistochemical analysis of TGHQ-derived protein adducts 2 h after dosing. Fischer 344 rats were dosed with either vehicle (PBS) (A) or TGHQ (20 µmol/kg, tail vein, iv) (B), albino guinea pigs (C and D), Golden Syrian hamsters (E and F), and B6C3F1 mice (G and H) were treated with either vehicle (PBS, C, E, and G) or TGHQ (200 µmol/kg, ic, D, F, and H). Slices of kidneys were preserved in zinc-buffered formalin, embedded in paraffin, sectioned (5 µm), immunostained with a 1:30 dilution of anti-2BrHQ-NAC antibodies, and counterstained with hematoxylin (9× magnification).
In rats and guinea pigs, the extent of immunostaining correlated with the subsequent region of necrosis ob-
served 19 h after dosing (7). Interestingly, despite the positive immunostaining in hamster kidney, this did not
1294 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Kleiner et al.
Figure 2. Immunohistochemical analysis of TGHQ-derived protein adducts 2 h after dosing. Fischer 344 rats were dosed with 20 µmol/kg TGHQ (tail vein, iv) (A). Albino guinea pigs (B) and Golden Syrian hamsters (C) were treated with 200 µmol/kg TGHQ (ic). Slices of kidneys were preserved in zinc-buffered formalin, embedded in paraffin, sectioned (5 µm), and immunostained with a 1:30 dilution of anti-2-BrHQ-NAC antibodies (90× magnification).
result in the development of necrotic tissue or significant decreases in renal function. Although in rats overt necrosis is not evident histologically by 2 h, increases in
the extent of urinary excretion of γ-GT occurs (Table 1). Thus, in rats and guinea pigs, covalent binding of TGHQ to proteins occurs in cells in the same region that
Species Differences in Quinone-Thioether Adducts A
B
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1295 C
D
Figure 3. Western blot analysis of TGHQ-derived protein adducts in kidney cytosolic fractions 2 h after dosing. B6C3F1 mice, Golden Syrian hamsters, and albino guinea pigs were dosed with 200 µmol/kg TGHQ (ic). Fischer 344 rats were dosed with 20 µmol/kg TGHQ (tail vein, iv). CB is Coomassie Blue stain; WB is Western blot immunostain. For illustrative purposes, rat vehicle control is shown on the right of panel D. Vehicle controls were also performed using the other species, and the immunostaining was also negative (data not shown). Proteins (250 µg/lane) were separated on a 10% SDS-PAGE reducing gel, transferred to nitrocellulose, and probed with anti-2-BrHQ-NAC antibodies (1:20 dilution) using an enhanced chemiluminescence detection system. Table 1. Species Differences in Urinary γ-Glutamyl Transpeptidase Activity 2 h after Treatment with TGHQa species
TGHQ treatment
activity (unitsb x 10-3/2 h)
B6C3F1 mouse B6C3F1 mouse Golden Syrian hamster Golden Syrian hamster albino guinea pig albino guinea pig Fischer 344 rat Fischer 344 rat
200 µmol/kg (ic) vehicle (ic) 200 µmol/kg (ic) vehicle (ic) 200 µmol/kg (ic) vehicle (ic) 20 µmol/kg (iv) vehicle (iv)
0.04 ( 0.02 0.05 ( 0.03 0.08 ( 0.02 0.16 ( 0.03 0.17 (0.14; 0.2) 0.10 (0.09; 0.10) 17.0 ( 5.65c 1.18 ( 0.12
a Data are expressed as means ( SE (n ) 3 in all species except guinea pig, where n ) 2). b One unit of activity is defined as that amount of enzyme that will produce 1 nmol of p-nitroaniline/min at 25 °C. c Statistically significant from vehicle control (P < 0.05) (Student’s t test).
subsequently develops overt tubular cell necrosis. These results confirm that TGHQ is activated to an electrophilic intermediate which covalently binds to protein at the site of injury. Western Blot Analysis. Western blot analysis was used to assess potential species differences in the qualitative nature of the covalently adducted proteins in renal subcellular fractions. Samples from at least two treated animals and one vehicle control animal were used for Western blot analysis. Western blots were performed at least twice to confirm the results. Representative samples are depicted in each figure for comparison across species. No immunostaining is seen in any of the untreated tissue samples (see below). For illustrative purposes, vehicle control samples are shown for Fischer 344 rats only, but the vehicle controls were assayed on the same Western blot for all the other species. In each case, no immunostaining is observed in any of the vehicle controls. Species Differences in Renal Subcellular Targets. Western blot analysis of renal subcellular fractions revealed numerous anti-2-BrHQ-NAC immunostaining
proteins in all species examined (summarized in Table 2). Although a variety of immunostained proteins are observed in the cytosolic fractions obtained from each species, no common adducted proteins were identified. The major immunostaining cytosolic proteins in the Fischer 344 rat exhibit MWs of 46 and 72 kDa (Figure 3). Other immunostaining proteins are detected in rat kidney cytosol at 37, 41, and 60 kDa (Figure 3). A 46 kDa immunopositive cytosolic protein is found in guinea pigs and a 72 kDa immunopositive protein in hamster cytosolic fractions (Figure 3). In the endoplasmic reticulum-enriched fractions, immunopositive proteins at 83 and 87 kDa are found in the rat (Figure 4). Neither hamsters nor guinea pigs exhibit major immunostaining proteins at either of these MWs. Immunostained proteins of 85 and 89 kDa are present in the endoplasmic reticulum-enriched fractions obtained from mice (Figure 4). Two major immunopositive proteins at 47 and 82 kDa are found in the plasma membrane-enriched fractions of rat kidney (Figure 5). It is possible the latter protein and the 83 kDa protein of the endoplasmic reticulum are the same, and occur in both fractions as a consequence of the enrichment protocol. Three immunopositive proteins (49, 69, and 110 kDa) in the guinea pig were unique to this species (Figure 5). In renal mitochondria (Figure 6), the most distinctive immunostaining protein is observed in Fischer 344 rats at approximately 34 kDa. This protein is also observed in guinea pigs and hamsters, although the intensity of immunostaining is lower in these species. A lowerintensity immunostaining protein at approximately 48 kDa is also observed in both B6C3F1 mice and Fischer 344 rat kidney mitochondria. In kidney nuclei (Figure 7), a protein of approximately 35 kDa is immunostained in Fischer 344 rats, and this protein is not detected in
1296 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
Kleiner et al.
Table 2. Species Differences in Immunochemical Detection of Covalent Protein Adducts of TGHQa subcellular fraction
B6C3F1 mouse
Golden Syrian hamster
albino guinea pig
Fischer 344 rat
renal cytosol renal ERb renal plasma membrane renal mitochondria renal nuclei urine hepatic cytosol hepatic ER
(36, 45, 50, 57, 61) 85, 89 (40-63, 101-178) 47 (48) NDc ND ND ND
49, 72, 86 (59, 111) 54, 62-195 66, 82, 94-142 (35, 50, 55, 78, 129) (56, 63, 96) ND ND ND
36, 46, 49, 73-120 (38, 41, 53, 57) 32, 45, 52, 67-150 49, 69, 110, 102-140 (34, 49, 74) (56) ND ND ND
46, 72 (37, 41, 60) 83, 87 (45, 65) 47, 82 34 (42, 48, 81) 35 (33, 51) 73 (49, 62, 78, 81, 89) ND ND
a Values represent estimated molecular masses (kilodaltons) of the average of two determinations (two individual animals) of anti-2BrHQ-NAC immunostaining (ECL detection). Western blots were scanned and analyzed using NIH Image software. Numbers in parentheses represent values with lower-intensity immunostaining. b ER, endoplasmic reticulum-enriched fractions. c ND, not detected.
A
B
C
D
Figure 4. Western blot analysis of TGHQ-derived protein adducts in kidney endoplasmic reticulum-enriched fractions 2 h after dosing. B6C3F1 mice, Golden Syrian hamsters, and albino guinea pigs were dosed with 200 µmol/kg TGHQ (ic). Fischer 344 rats were dosed with 20 µmol/kg TGHQ (tail vein, iv). CB is Coomassie Blue stain; WB is Western blot immunostain. For illustrative purposes, rat vehicle control is shown on the right of panel D. Vehicle controls were also performed using the other species, and the immunostaining was also negative (data not shown). Proteins (250 µg/lane) were separated on a 10% SDS-PAGE reducing gel, transferred to nitrocellulose, and probed with anti-2-BrHQ-NAC antibodies (1:20 dilution) using an enhanced chemiluminescence detection system.
any of the other species. In summary, it is clear that the pattern of immunostained proteins present in the kidney of TGHQ-treated animals is unique to each species. This “fingerprint” may be important in determining susceptibility to toxicity. Quinone-Thioether-Adducted Proteins in Urine. To compare species differences in TGHQ-derived renal protein adducts shed into urine as a result of cytotoxicity, Western blots were performed on urine obtained from each species 2 h after dosing with TGHQ (Figure 8). At this time point, no overt cytotoxicity was observed. The predominant immunostaining protein in Fischer 344 rat urine migrates at approximately 73 kDa. Lower-intensity immunostaining proteins are also detected in Fischer 344 rat urine, at approximately 49, 62, 78, 81, and 89 kDa. Immunostaining is not detected in B6C3F1 mouse, guinea pig, or hamster urine. To preliminarily characterize the 73 kDa quinonethioether binding protein in Fischer 344 rat urine, an authentic γ-GT standard (20 µg) (>99.9% pure bovine γ-GT, Calbiochem) was loaded onto the gel for direct
comparison. Under reducing conditions, authentic γ-GT migrates at approximately 73 kDa. Thus, the 73 kDa quinone-thioether binding protein that is detected in Fischer 344 rat urine may represent γ-GT, although positive identification requires more rigorous analysis. In support of this suggestion, Fischer 344 rats displayed significant elevations (Table 1) in urinary γ-GT, which is an early indicator of quinone-thioether-mediated nephrotoxicity in rats. Quinone-Thioether-Adducted Proteins in Liver Cytosol and Microsomes. There were no immunostaining proteins detected in liver cytosol or endoplasmic reticulum-enriched fractions in any of the species studied (data not shown).
Discussion The immunohistological localization of TGHQ-derived protein adducts in Fischer 344 rats and albino guinea pigs 2 h after dosing (Figures 1 and 2) correlates with
Species Differences in Quinone-Thioether Adducts A
B
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1297 C
D
Figure 5. Western blot analysis of TGHQ-derived protein adducts in kidney plasma membrane-enriched fractions 2 h after dosing. B6C3F1 mice, Golden Syrian hamsters, and albino guinea pigs were dosed with 200 µmol/kg TGHQ (ic). Fischer 344 rats were dosed with 20 µmol/kg TGHQ (tail vein, iv). CB is Coomassie Blue stain; WB is Western blot immunostain. For illustrative purposes, rat vehicle control is shown on the right of panel D. Vehicle controls were also performed using the other species, and the immunostaining was also negative (data not shown). Proteins (250 µg/lane) were separated on a 10% SDS-PAGE reducing gel, transferred to nitrocellulose, and probed with anti-2-BrHQ-NAC antibodies (1:20 dilution) using an enhanced chemiluminescence detection system. A
B
C
D
Figure 6. Western blot analysis of TGHQ-derived protein adducts in kidney mitochondria-enriched fractions 2 h after dosing. B6C3F1 mice, Golden Syrian hamsters, and albino guinea pigs were dosed with 200 µmol/kg TGHQ (ic). Fischer 344 rats were dosed with 20 µmol/kg TGHQ (tail vein, iv). CB is Coomassie Blue stain; WB is Western blot immunostain. For illustrative purposes, rat vehicle control is shown on the right of panel D. Vehicle controls were also performed using the other species, and the immunostaining was also negative (data not shown). Proteins (250 µg/lane) were separated on a 10% SDS-PAGE reducing gel, transferred to nitrocellulose, and probed with anti-2-BrHQ-NAC antibodies (1:20 dilution) using an enhanced chemiluminescence detection system.
the region of necrosis subsequently observed 19 h after dosing (7). Thus, immunostaining reveals the presence of TGHQ-derived protein adducts prior to overt signs of renal necrosis. No adducted proteins were observed
inregions of the rat kidney where necrosis does not occur (such as the papillae or the glomeruli), and adducted proteins were not observed in nontarget tissue (liver) of any species.
1298 Chem. Res. Toxicol., Vol. 11, No. 11, 1998 A
Kleiner et al. C
B
D
Figure 7. Western blot analysis of TGHQ-derived protein adducts in kidney nuclei-enriched fractions 2 h after dosing. B6C3F1 mice, Golden Syrian hamsters, and albino guinea pigs were dosed with 200 µmol/kg TGHQ (ic). Fischer 344 rats were dosed with 20 µmol/kg TGHQ (tail vein, iv). CB is Coomassie Blue stain; WB is Western blot immunostain. For illustrative purposes, rat vehicle control is shown on the right of panel D. Vehicle controls were also performed using the other species, and the immunostaining was also negative (data not shown). Proteins (250 µg/lane) were separated on a 10% SDS-PAGE reducing gel, transferred to nitrocellulose, and probed with anti-2-BrHQ-NAC antibodies (1:20 dilution) using an enhanced chemiluminescence detection system. A
B
C
D
E
Figure 8. Western blot analysis of TGHQ-derived protein adducts excreted in urine 2 h after dosing. B6C3F1 mice, Golden Syrian hamsters, and albino guinea pigs were dosed with 200 µmol/kg TGHQ (ic). Fischer 344 rats were dosed with 20 µmol/kg TGHQ (tail vein, iv). CB is Coomassie Blue stain; WB is Western blot immunostain. For illustrative purposes, rat vehicle control is shown on the right of panel E. Vehicle controls were also performed using the other species, and the immunostaining was also negative (data not shown). Proteins (250 µg/lane) were separated on a 10% SDS-PAGE reducing gel, transferred to nitrocellulose, and probed with anti-2-BrHQ-NAC antibodies (1:20 dilution) using an enhanced chemiluminescence detection system. + indicates treatment with TGHQ; - indicates vehicle control. The lane marked γ-GT indicates Coomassie Blue stain of pure (>99.9%) bovine γ-GT (20 µg).
The hamster does not develop nephrotoxicity when exposed to TGHQ, yet immunohistochemical staining revealed TGHQ-derived protein adducts in approximately the same region of the kidney as in rats (Figures 1 and
2). This indicates that not only are hamster kidneys exposed to potentially toxic electrophilic metabolites of TGHQ but also these metabolites form covalent adducts with proteins in regions of the kidney that progress to
Species Differences in Quinone-Thioether Adducts
necrosis in other species. The hamster therefore represents an intriguing model with which to examine the role of covalent adducts in quinone-thioether-induced nephrotoxicity. One explanation for the lack of nephrotoxicity in hamsters, despite the presence of covalently adducted proteins, lies in the nature of the adducted proteins. Thus, the pattern of adducted proteins in hamster kidney, revealed by Western blotting, is substantially different from the patterns in either rats or guinea pigs (Figures 3-7). In addition, either these adducted proteins represent benign lesions to noncritical proteins or the damaged proteins are repaired more efficiently than in rats and guinea pigs. Identification of the adducted proteins, and a determination of the rate at which the adducted proteins are removed and/or repaired, will help determine which of these alternative explanations is valid. Immunohistochemical analysis revealed only faint staining in kidneys of B6C3F1 mice (Figure 1), a species resistant to the toxic effects of TGHQ. Although Western analysis, probed using enhanced chemiluminescence, a far more sensitive technique than immunohistochemical analysis, did reveal the presence of adducted proteins in mouse kidney (Figures 3-7), the time required to develop the staining indicates that these proteins are present in comparatively minor amounts. Species differences were seen in the pattern of TGHQderived covalent protein adducts, determined by Western analysis (Figures 3-8). Consistent with the immunohistochemical analysis (data not shown), no immunostaining is detected in cytosol (Figure 8) or endoplasmic reticulum-enriched fractions (data not shown) of liver, a nontarget organ. Although there are numerous TGHQderived protein adducts in both sensitive (Fischer 344 rats and guinea pigs) and resistant (hamsters and B6C3F1 mice) species (Table 2), the most striking qualitative difference seen across species was found at approximately 34-35 kDa in rat kidney mitochondria and nuclei, and at approximately 73 kDa in rat urine. Although mitochondria are not primary targets of quinone-thioether-mediated nephrotoxicity (19, 20), covalent binding to renal mitochondrial proteins in Fischer 344 rats does occur, and the pattern of binding is different from that seen in nonsensitive species (B6C3F1 mice and hamsters) (Figure 6). Whether the 34-35 kDa quinone-thioether binding protein in Fischer 344 rat kidney nuclei and mitochondria is the same remains to be determined. Linker histone H1 comigrates with these proteins on 13.5% SDS-PAGE gels (data not shown). However, mitochondrial DNA is not associated with histone proteins. The synthetic estrogen diethylstilbestrol, a renal carcinogen bioactivated to a reactive quinone, covalently binds both in vivo in hamster kidney nuclei and in vitro to histone and nonhistone (∼28, 33, 37, and 56 kDa) nuclear proteins (18). In vitro, [3H]diethylstilbestrolquinone also forms adducts with pure RNA and DNA polymerases (18). Immunoprecipitation with anti-histone antibodies and amino acid microsequence analysis will be used to determine whether the 35 kDa nuclear quinone-thioether binding protein is a histone. Nuclear protein binding by quinone-thioethers is of special interest since karyolysis and karyorrhexis occur 2 h after treatment of male Fischer 344 rats with BrBGHQ (30 µmol/kg, iv), as observed by electron microscopy (19). This is followed by dissolution of the nuclear membrane, and random DNA fragmentation (19).
Chem. Res. Toxicol., Vol. 11, No. 11, 1998 1299
Consistent with the in vivo findings, treatment of renal epithelial cells (LLC-PK1) with 2-Br6GHQ and BrBGHQ results in the rapid formation of DNA single-strand breaks (21-23). Concomitant with DNA damage, growth arrest occurs rapidly as evidenced by a decrease in DNA synthesis (22), increases in the expression of the growth arrest and DNA damage inducible gene gadd153 (22, 23), and decreases in histone mRNA expression (24). Growth arrest probably occurs in an attempt to repair DNA damage prior to cell division. Consistent with the in vitro studies, gadd153 is upregulated and histone mRNA downregulated 2-4 h after TGHQ (7.5 µmol/kg, iv) administration to rats (25). However, growth arrest in vivo is followed by a wave of cell proliferation, 24-96 h after dosing (5). The region of cell proliferation correlates with the eventual location of renal tumors following longterm HQ administration to rats (5). Covalent modification of histones by quinone-thioethers, causing changes in chromatin structure and function, especially alterations in gene expression, may contribute to HQ-induced nephrocarcinogenesis. Immunochemical analysis of urine from rats treated with TGHQ identified several immunostaining proteins. The ∼73 kDa rat urinary quinone-thioether binding protein is probably γ-GT, since an authentic γ-GT standard comigrated with this protein on 10% SDSPAGE gels (Figure 8), and there is significant urinary excretion of γ-GT in rats (Table 1). Furthermore, TGHQ is a substrate for γ-GT (7) and inhibits the enzyme in LLC-PK1 cells (26). TGHQ therefore appears to be a “suicide” substrate for γ-GT, and the binding of adducted protein in urine with a MW identical to that of γ-GT is consistent with this view. In summary, anti-2-BrHQ-NAC antibodies recognized several TGHQ-derived covalent protein adducts in regions of the rat kidney that subsequently undergo necrosis. In this manner, the covalent adducts serve as a marker for cells that will eventually die. In contrast, the presence of covalently adducted proteins in hamster kidney, in cells which do not progress to necrosis, provides an excellent model with which to determine the toxicological relevance of these lesions. The future identification of these proteins will be essential to understanding the role of covalent protein binding in quinone-thioether-mediated toxicities. Moreover, because quinone-thioethers have also been implicated in a number of human disease(s) (27, 28), including Parkinson’s disease (29-31), alcoholism (32, 33), and cancer (5, 34-36), the ability to identify proteins modified during the pathogenesis of these diseases may provide insight into the disease process.
Acknowledgment. This work was supported in part by an award from the National Institute of General Medical Sciences [GM39338 (S.S.L.)]. H.E.K. was a recipient of the CIBA-Geigy Graduate Student Fellowship sponsored by the Society of Toxicology. We also thank Drs. Irma Gimenez de Conti for her assistance with the immunohistochemical staining and Dennis Johnston for his help with the analysis of Western blots using NIH image analysis under the support of Center Grant ES 07784.
References (1) Kari, F. W. (1989) Toxicology and carcinogenesis studies of hydroquinone in F344/N rats and B6C3F1 mice (gavage studies).
1300 Chem. Res. Toxicol., Vol. 11, No. 11, 1998
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10) (11)
(12)
(13) (14) (15) (16)
(17)
(18) (19)
National Toxicology Program Technical Report, p 366, U.S. Department of Health and Human Services, U.S. Public Health Service, National Institutes of Health. Kari, F. W., Bucher, J., Eustis, S. L., Haseman, J. K., and Huff, J. E. (1992) Toxicity and carcinogenicity of hydroquinone in F344/N rats and B6C3F1 mice. Fundam. Chem. Toxicol. 30, 737747. Shibata, M.-A., Hirose, M., Tanaka, H., Asakawa, E., Shirai, T., and Ito, N. (1991) Induction of renal cell tumors in rats and mice, and enhancement of hepatocellular tumor development in mice after long-term hydroquinone treatment. Jpn. J. Cancer Res. 82, 1211-1219. Lau, S. S., Hill, B. A., Highet, R. J., and Monks, T. J. (1988) Sequential oxidation and glutathione addition to 1,4-benzoquinone: correlation of toxicity with increased glutathione substitution. Mol. Pharmacol. 34, 829-836. Peters, M. C. G., Jones, T. W., Monks, T. J., and Lau, S. S. (1997) Cytotoxicity and cell-proliferation induced by the nephrocarcinogen hydroquinone and its nephrotoxic metabolite 2,3,5-tris(glutathion-S-yl)hydroquinone. Carcinogenesis 18, 2393-2401. Hill, B. A., Kleiner, H. E., Ryan, E. A., Dulik, D. M., Monks, T. J., and Lau, S. S. (1993) Identification of multi-S-substituted conjugates of hydroquinone by HPLC-coulometric electrode array analysis and mass spectroscopy. Chem. Res. Toxicol. 6, 459-469. Lau, S. S., Kleiner, H. E., and Monks, T. J. (1995) Metabolism as a determinant of species susceptibility to 2,3,5-(triglutathion-Syl)hydroquinone-mediated nephrotoxicity. The role of N-acetylation and N-deacetylation. Drug Metab. Dispos. 23, 1136-1142. Satoh, H., Fukuda, Y., Anderson, D. K., Ferrans, V. J., Gillette, J. R., and Pohl, L. R. (1985) Immunological studies on the mechanism of halothane-induced hepatotoxicity: immunohistochemical evidence of trifluoroacetylated hepatocytes. J. Pharmacol. Exp. Ther. 233, 857-862. Heijink, E., De Matteis, F., Gibbs, A. H., Davies, A., and White, I. N. H. (1993) Metabolic activation of halothane to neoantigens in C57Bl/10 mice: immunochemical studies. Eur. J. Pharmacol. 248, 15-25. Bartolone, J. B., Cohen, S. D., and Khairallah, E. A. (1989) Immunohistochemical localization of acetaminophen-bound liver proteins. Fundam. Appl. Toxicol. 13, 859-862. Salminen, W. F., Jr., Roberts, S. M., Pumford, N. R., and Hinson, J. A. (1998) Immunochemical comparison of 3′-hydroxyacetanilide and acetaminopehn binding in mouse liver. Drug. Metab. Dispos. 26, 267-271. Hayden, P. J., Ichimura, T., McCann, D. J., Pohl, L. R., and Stevens, J. L. (1991) Detection of cysteine conjugate metabolite adduct formation with specific mitochondrial proteins using antibodies raised against halothane metabolite adducts. J. Biol. Chem. 266, 18415-18418. Kleiner, H. E., Rivera, M. I., Pumford, N. R., Monks, T. J., and Lau, S. S. (1998) Immunochemical detection of quinol-thioether derived protein adducts. Chem. Res. Toxicol. 11, 1283-1290. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc. Natl. Acad. Sci. U.S.A. 76, 4350-4354. Kenna, J. G., Neuberger, J., and Williams, R. (1987) Identification by immunoblotting of three halothane-induced liver microsomal polypeptide antigens recognized by antibodies in sera from patients with halothane-associated hepatitis. J. Pharmacol. Exp. Ther. 242, 733-740. Roy, D., Pathak, D. N., and Palangat, M.L (1995) In vivo binding of diethylstilbestrol to nuclear proteins of kidneys of Syrian hamsters. Cancer Lett. 90, 215-224. Rivera, M. I., Jones, T. W., Lau, S. S., and Monks, T. J. (1994) Early morphological and biochemical changes during 2-bromo(diglutathion-S-yl)hydroquinone-induced nephrotoxicity. Toxicol. Appl. Pharmacol. 128, 239-250.
Kleiner et al. (20) Hill, B. A., Monks, T. J., and Lau S. S. (1992) The effects of 2,3,5(triglutathion-S-yl)hydroquinone on renal mitochondrial respiratory function in vivo and in vitro: possible role in cytotoxicity. Toxicol. Appl. Pharmacol. 117, 165-171. (21) Mertens, J. J. W. M., Gibson, N. W., Lau, S. S., and Monks, T. J. (1995) Reactive oxygen species and DNA damage in 2-bromo(glutathion-S-yl)hydroquinone-mediated cytotoxicity. Arch. Biochem. Biophys. 320, 51-58. (22) Jeong, J. K., Stevens, J. L., Lau, S. S., and Monks, T. J. (1996) Quinone-thioether-mediated DNA damage, growth arrest, and gadd153 expression in renal proximal tubular epithelial cells. Mol. Pharmacol. 50, 592-598. (23) Jeong, J. J., Dybing, E., Søderlund, E., Brunborg, G., Holme, J. A., Lau, S. S., and Monks, T. J. (1997) DNA damage, gadd153 expression, and cytotoxicity in plateau-phase renal proximal tubular epithelial cells treated with a quinol-thioether. Arch. Biochem. Biophys. 341, 300-308. (24) Jeong, J. K., Huang, Q., Lau, S. S., and Monks, T. J. (1997) The response of renal tubular epithelial cells to physiologically and chemically induced growth arrest. J. Biol. Chem. 272, 7511-7518. (25) Peters, M. M. C. G., Monks, T. J., and Lau, S. S. (1996) Stress gene and growth related gene expression following acute renal injury. Fundam. Appl. Toxicol. 30, 331 (abstract). (26) Hill, B. A., Lo, H.-H., Monks, T. J., and Lau, S. S. (1991) The role of γ-glutamyltranspeptidase in hydroquinone-glutathione conjugate mediated nephrotoxicity. Adv. Exp. Med. Biol. 283, 749751. (27) Monks, T. J., and Lau, S. S. (1997) Biological reactivity of polyphenolic-glutathione conjugates. Chem. Res. Toxicol. 10, 1296-1313. (28) Monks, T. J., and Lau, S. S. (1998) The pharmacology and toxicology of polyphenolic-glutathione conjugates. Annu. Rev. Pharmacol. Toxicol. 38, 229-255. (29) Shen, X. M., and Dryhurst, G. (1996) Further insights into the influence of L-cysteine on the oxidation chemistry of dopamine: reaction pathways of potential relevance to Parkinson’s disease. Chem. Res. Toxicol. 9, 751-763. (30) Shen, X. M., Xia, B., Wrona, M. Z., and Dryhurst, G. (1996) Synthesis, redox properties, in vivo formation, and neurobehavioral effects of N-acetylcysteinyl conjugates of dopamine: possible metabolites of relevance to Parkinson’s disease. Chem. Res. Toxicol. 9, 1117-1126. (31) Li, H., and Dryhurst, G. (1997) Irreversible inhibition of mitochondrial complex I by 7-(2-aminoethyl)-3,4-dihydro-5-hydroxy2H-1,4-benzothiazine-3-carboxylic acid (DHBT-1): a putative nigral endotoxin of relevance to Parkinson’s disease. J. Neurochem. 69, 1530-1541. (32) Han, Q. P., and Dryhurst, G. (1996) Influence of glutathione on the oxidation of 1-methyl-6-hydroxy-1,2,3,4-tetrahydro-β-carboline: chemistry of potential relevance to the addictive and neurodegenerative consequences of ethanol use. J. Med. Chem. 39, 1494-1508. (33) Wrona, M. Z., Waskiewicz, J., Han, Q. P., Han, J., Li, H., and Dryhurst, G. (1997) Putative oxidative metabolites of 1-methyl6-hydroxy-1,2,3,4-tetrahydro-β-carboline of potential relevance to the addictive and neurodegenerative consequences of ethanol abuse. Alcohol 14, 213-223. (34) Peters, M. M. C. G., Rivera, M. I., Jones, T. W., Monks, T. J., and Lau, S. S. (1996) Glutathione conjugates of tert-butyl hydroquinone, a metabolite of the urinary tract tumor promoter 3-tertbutylhydroxyanisole, are toxic to kidney and bladder. Cancer Res. 56, 1006-1011. (35) Butterworth, M., Lau, S. S., and Monks, T. J. (1998) 2-Hydroxy4-glutathion-S-yl-17β-estradiol and 2-hydroxy-1-glutathion-S-yl17β-estradiol produce oxidative stress and renal toxicity in an animal model of 17β-estradiol-mediated nephrocarcinogenicity. Carcinogenesis 19, 133-139. (36) Lau, S. S., Monks, T. J., Everitt, J. I., and Walker, C. L. (1998) A nephrotoxic metabolite of hydroquinone induces renal cell tumorigenesis in Eker rats. Toxicol. Sci. 42, 317 (abstract).
TX9801357
