Characterization of tert-Butyl Alcohol Binding to {alpha}2u-Globulin in F-344 Rats

T. M. Williams and S. J. Borghoff,1

CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, North Carolina 27709

Received December 29, 2000; accepted May 7, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
tert-Butyl alcohol (TBA) is widely used in the manufacturing of certain perfumes, cosmetics, drugs, paint removers, methyl tert-butyl ether (MTBE), and industrial solvents. In both rodents and humans, TBA is a major metabolite of MTBE, an oxygenated fuel additive. Chronic TBA exposure causes protein droplet nephropathy, {alpha}2u-globulin ({alpha}2u) accumulation, renal cell proliferation, and with chronic exposure, renal tumors in male, but not female, rats. These effects suggest an {alpha}2u-mediated mechanism for renal tumors. The objective of the present study was to determine whether TBA or its metabolites bind to {alpha}2u. Mature male and female F-344 rats were administered a single gavage dose of 500 mg/kg TBA, 500 mg/kg 14C-TBA, or corn oil. TBA equivalents/gram or ml of tissue in the male rat kidney, liver, and blood were higher than the levels measured in female rat tissue 12 h after 14C-TBA administration. Gel filtration and anion-exchange chromatography demonstrated that 14C-TBA–derived radioactivity co-eluted with {alpha}2u from male kidney cytosol. Protein dialysis studies demonstrated that the interaction between 14C-TBA–derived radioactivity and {alpha}2u was reversible. Incubations of the low-molecular-weight protein fraction (LMWPF) isolated from 14C-TBA–treated male rat kidneys with d-limonene oxide (a chemical with a high affinity to {alpha}2u) demonstrated that 14C-TBA–derived radioactivity was displaced. Gas chromatography-mass spectrometry analysis confirmed that TBA was present in this LMWPF fraction. These results demonstrate that TBA interacts with {alpha}2u, which explains the accumulation of {alpha}2u in the male rat kidney following TBA exposure.

Key Words: tert-butyl alcohol (TBA); {alpha}2u-globulin ({alpha}2u) nephropathy; male rats; protein binding.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
tert-Butyl alcohol (TBA) is widely used in the manufacturing of certain perfumes, cosmetics, drugs, paint removers, methyl tert-butyl ether (MTBE), and industrial solvents (Budavari, 1989Go; Cirvello et al., 1995Go; Lington and Bevan, 1994Go; NTP, 1995Go). It is also an indirect food additive when it is used in the preparation and application of coatings for paperboard. Potential human exposure to TBA is high due to its diverse uses.

Chronic exposure to TBA in drinking water resulted in an increase in renal tubule adenomas and carcinomas in male, but not female, rats (Cirvello et al., 1995Go; NTP, 1995Go). Although one recent study demonstrated that TBA was mutagenic in the Ames assay using the TA102 strain (Williams-Hill et al., 1999Go), in all other systems tested, TBA was found to be negative (McGregor et al., 1988Go; NTP, 1997Go; Zeiger et al., 1987Go). This suggests that TBA most likely operates through a nongenotoxic mode of action to induce tumors in rodents.

In a 13-week study in which F-344 rats were administered from 0 to 4% TBA in the drinking water, TBA caused protein droplet accumulation and mineralization in male rat kidneys (Lindamood et al., 1992Go; Takahashi et al., 1993Go). These features are characteristic of {alpha}2u-globulin nephropathy ({alpha}2u-N), a syndrome considered unique to male rats, which is characterized by the accumulation of {alpha}2u-globulin ({alpha}2u), a low-molecular-weight protein, in the form of protein droplets within the renal proximal tubule (Borghoff et al., 1990Go; Swenberg and Lehman-McKeeman, 1999Go). In this study analyses were not performed to definitively confirm the presence of {alpha}2u in the protein droplets. However, neither F-344 female rats nor B6C3F1 mice of either sex developed protein droplets, which is an observation consistent with {alpha}2u accumulation.

To further investigate the ability of TBA to cause {alpha}2u-N in male rats, F-344 rats were exposed, via inhalation, to 0, 250, 450, and 1750 ppm TBA 6 h/day for 10 consecutive days. Protein droplets observed in male rat kidneys exposed to TBA were found to be immunoreactive for {alpha}2u (Borghoff et al., 2001Go). TBA also increased the concentration of {alpha}2u and enhanced cell proliferation in only male rat kidneys. The increase in {alpha}2u concentration was approximately a 1.6-fold increase over control, whereas the increase demonstrated following administration of 2,2,4-trimethylpentane (TMP) was approximately 3-fold. The induction of {alpha}2u-nephropathy following exposure to TBA was mild compared to what has previously been reported for 1,4-dichlorobenzene (1,4-DCB) (Charbonneau et al., 1989Go), TMP (Lock et al., 1987Go), or d-limonene (Lehman-Mckeeman et al., 1989Go). Following 8 consecutive days of TBA exposure, TBA was also found to be retained longer in the kidney of male rats compared to female rats.

Following exposure to chemicals that cause {alpha}2u-N, either the parent compound and/or a metabolite binds to {alpha}2u in a reversible, noncovalent manner. The formation of this chemical-{alpha}2u complex renders the protein resistant to hydrolysis (Lehman-McKeeman et al., 1990Go). As such, {alpha}2u accumulates in proximal tubule epithelial cells leading to necrosis and enhanced renal cell proliferation (Borghoff et al., 1990Go; Hard et al., 1993Go; Swenberg and Lehman-McKeeman, 1999Go).

Since TBA administration (either orally or via inhalation) to male rats causes {alpha}2u accumulation in the kidney, we hypothesized that TBA, or its metabolites, interacts with {alpha}2u, resulting in retention of the protein. The objective of the present study was to determine if TBA or one of its metabolites, 2-methyl-1,2-propanediol (MPD) or {alpha}-hydroxyisobutyric acid (HBA), bind to {alpha}2u. Since chemical binding to {alpha}2u and the formation of a chemical-{alpha}2u complex is critical to the development of {alpha}2u-globulin nephropathy, demonstration of binding would provide additional information to support the classification of TBA as an inducer of {alpha}2u-globulin nephropathy. If the TBA-induced kidney tumors in male rats is mediated through the ability of TBA to induce {alpha}2u-globulin nephropathy, and not through any other mode of action, then this tumor data will not be used as weight of evidence in the human hazard identification of TBA (U.S. EPA, 1991Go). These data are critical for human health risk assessment for this chemical.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
tert-Butyl alcohol (CAS No. 75–65–0) was obtained from Sigma Chemical Company (St. Louis, MO), 14C-tert-Butyl alcohol (50 mCi/mmol) was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO), and d-limonene oxide was obtained from Aldrich Chemical Co. (Milwaukee, WI). All chemicals were >97% pure.

Animals.
Nine-week-old male and female Fischer 344 (F-344) rats were obtained from Charles River Laboratories (Raleigh, NC) and allowed to acclimate for 12–14 days prior to use. Rats were randomized by weight into treatment groups and housed individually in polycarbonate cages. The temperature was controlled to 20 ± 1°C and relative humidity to 30–70%. Environmental parameters were recorded every 30 min for a 24-h period by an Infinity Building Automation System (Andover Controls Corporation, Andover, MA). Rats received pelleted food (NIH-07, Zeigler Bros., Gardners, PA) and deionized, filter-purified water, ad libitum. A 12-h light-dark cycle was maintained. These studies were performed under National Research Council guidelines (1996) for animal use and were approved by the Institutional Animal Care and Use Committee of CIIT.

TBA administration.
Rats were dosed once with 500 mg/kg TBA, 500 mg/kg 14C-TBA, or vehicle (corn oil) by gavage (n = 4). This dose was selected based on a previous report in which 5 mg/ml TBA in drinking water (equivalent to ~ 420 mg/kg) produced an accumulation of protein droplets in the kidney in male F-344 rats following 13 weeks of administration (NTP, 1995Go). The selection of one dose of TBA, which was known to cause protein droplet accumulation, was appropriate since our objective was to evaluate the capability of TBA or a metabolite to bind to {alpha}2u. Dosing solutions were calculated based on 3-ml/kg body weight. TBA concentrations in the dosing solution was verified by headspace analysis, using a Model 7694 headspace autosampler (Hewlett Packard, Avondale, PA) connected to a Hewlett Packard 5890 Series II gas chromatograph fitted with a capillary injection port and a flame ionization detector.

TBA equivalents in tissues and urine.
Six male and 6 female rats were housed in metabolic cages for 16 h prior to dosing, and urine was collected on dry ice. Rats were dosed with 500 mg/kg 14C-TBA or corn oil and euthanized by decapitation 12 h following dosing. A 1-ml aliquot of whole-trunk blood was digested in 2 x (w/v) tetraethylammonium hydroxide (TEAH). The liver and kidneys were removed and weighed. Kidneys were minced and homogenized in 3 x (w/v) 0.1 M phosphate buffer (pH 7.4) using an Ultra-Turrax homogenizer (Tekmar Co., Cincinnati, OH). Liver tissue and kidney homogenate were digested in 2 x (w/v) and 3 x (w/v) TEAH, respectively, for 30 h. Aliquots of TEAH-digested tissues, blood (1 ml), kidney (0.2 ml), and liver (2 ml), were neutralized by the addition of 0.2 ml HCl/ml digested tissue. These samples were then decolorized by the addition of 0.5 ml 35% H2O2/ml digested tissue for 72 h at room temperature. Following the addition of EcoLume® scintillation cocktail (ICN Biomedicals, Costa Mesa, CA), the samples were placed in the dark for 16 h at room temperature to decrease quenching. Aliquots of pre- and postadministration urine (0.1 ml) from 14C-TBA–treated rats were added to 7 ml of scintillation cocktail. All samples were counted for 5 min using a Tri-Carb 1900CA liquid scintillation analyzer (Parkard Instruments Co., Meriden, CT). Results were expressed as µmols of TBA equivalents/g of tissue or ml of blood or urine.

The Student's t test was used to compare the µmols of TBA equivalents/g of tissue or ml of blood or urine in male and female rats. The statistical significance was defined at p <= 0.05.

Preparation of kidney cytosol.
Four additional male and female rats were gavaged once with 500 mg/kg TBA, 500 mg/kg 14C-TBA, or corn oil. Rats were euthanized by decapitation 12 h following dosing. Kidneys were removed and weighed, then minced and homogenized in 3 x (w/v) 0.1 M phosphate buffer (pH 7.4), using an Ultra-Turrax homogenizer and frozen at –80°C. Kidney cytosol was prepared by ultracentrifugation of thawed kidney homogenate (116,000 x g, 1 h), and the cytosol was stored at –80°C.

Low-molecular-weight protein fraction (LMWPF).
Kidney cytosol protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL) based on the method of Smith et al. (1985) using bovine serum albumin as a standard. Kidney cytosol, 20 mg of total protein, was applied to a gel filtration column (1.5 cm id x 90 cm) at 4°C with Sephadex G-75 packing (Amersham Pharmacia Biotech, Piscataway, NJ) and eluted with 20 mM Tris buffer (pH 7.8) at 18 ml/h. The eluant was monitored at 280 nm and collected in 1.5-ml fractions. The column was previously calibrated with blue dextran (2000 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), purified {alpha}2u as described below (18.7 kDa), and ribonuclease A (13.7 kDa). Scintillation cocktail was added to the low-molecular-weight protein fractions (1.5 ml), eluted from the column, and counted using a liquid scintillation analyzer, as described above. Gel filtration analyses were performed in triplicate. Additional LMWPF from control and TBA-treated rats were pooled and concentrated using an Amicon Ultrafiltration System (Beverly, MA) with Diaflo ultrafiltration membranes (YM10, 43 mm). The concentrated LMWPF was stored at –20°C prior to protein dialysis, fast-protein liquid chromatography (FPLC) analysis, and gas chromatography-mass spectrometry (GC-MS) analysis.

Purified urinary {alpha}2u-globulin.
Urine was collected from 16 untreated male rats housed in metabolic cages from 3:00 P.M. to 9:00 A.M. for 20 days over a 4-week period. Urine was collected daily, pooled, and centrifuged (500 x g, 15 min). The resulting supernatant was frozen at –80°C. Following urine collection, the frozen, pooled supernatant was thawed on ice, and the urinary proteins were precipitated with 85% ammonium sulfate and resuspended in 2-pellet volumes of distilled water as described by Kurtz et al. (1976). Total urinary protein was concentrated and dialyzed on an Amicon Ultrafiltration System as described above. The dialysate was applied to a Sephadex G-75 gel filtration column to collect the LMWPF, and {alpha}2u was isolated by FPLC as described by Borghoff and Lagarde (1993). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were used to assess the purity of the isolated {alpha}2u, as described by Borghoff and Lagarde (1993).

Measurement of {alpha}2u.
The concentration of {alpha}2u was measured in kidney cytosol from male rat kidneys using an enzyme-linked immunosorbent assay (ELISA) described by Borghoff et al. (1992). Analyses were performed in quadruplicate. A mouse monoclonal antibody raised toward purified rat urinary {alpha}2u was used (prepared by Hazelton Biotechnologies Co., Vienna, VA). The monoclonal antibody directed against {alpha}2u did not show any cross-reactivity with other proteins in kidney cytosol, using SDS-PAGE and Western blot analysis (unpublished observation). Kidney cytosol from male F-344 rats gavaged with 500-mg/kg trimethylpentane, a strong inducer of {alpha}2u-globulin nephropathy, was used as a positive control (Prescott-Mathews et al., 1997Go).

FPLC analysis.
The LMWPF was analyzed at room temperature using a Pharmacia FPLC equipped with a Waters DEAE ion-exchange column (7.5 mm x 7.5 cm, 1000 Å), an LCC-500 gradient controller, and a 2-pump system. Buffer A consisted of 20 mM Tris (pH 8.2) and buffer B consisted of 20 mM Tris buffer with 0.5 N NaCl (pH 8.2). The gradient was held at 0% B for 5 min and then increased to 5% B in 5 min, to 10% B in 15 min, to 15% B in 20 min, to 20% B in 15 min, and to 100% B in 10 min. The total run time was 70 min with a flow rate of 1 ml/min. The {alpha}2u eluted into 2 distinct peaks between 12 and 15% B. The eluant was monitored at 280 nm and collected in 1-ml fractions. Fractions were dissolved in 6-ml scintillation cocktail and counted as previously described. FPLC analyses were performed in triplicate.

Following FPLC analysis of the LMWPF from the kidneys of 14C-TBA–treated rats, the LMWPF from the kidneys of TBA-treated rats was analyzed by the same method described above. The 2 FPLC peaks that corresponded to fractions containing 14C-TBA–derived radioactivity were dialyzed with deionized water for 24 h at 4°C in a Spectra/Por membrane with a molecular weight cut-off between 13,000 and 15,000 (Spectrum Medical Industries, Inc., Houston, TX). The dialyzed samples were lyophilized overnight. Western blot analysis was performed on a semidry electrophoretic transfer system (PhastTransfer, Pharmacia/LKB) using an anti-{alpha}2u antibody as described by Borghoff and Lagarde (1993) to determine whether the 14C-TBA–derived radioactivity co-eluted with {alpha}2u. Analyses were performed in triplicate.

GC-MS analysis.
Aliquots (0.1 ml) of the LMWPF were sealed in 7-ml glass vials and heated at 65°C for 15 min. The headspace (0.1-ml) was analyzed for TBA using a Hewlett Packard 5989B mass spectrometer coupled to an HP Series II Plus 5890 gas chromatograph equipped with a DB-WAX column (0.32 mm id, 30 m; J & W Scientific, Inc., Folsom, CA). The headspace was manually injected on-column. The on-column inlet was operated in a constant-flow mode (1.5 psi, He) with an initial temperature of 225°C. The column was held at 25°C for 1.50 min following injection, then raised at 15°C/min to 150°C and held for 0.3 min, and then raised at 50°C/min to 200°C and held for 1.0 min. The mass spectrometer was operated in the electron ionization mode (EI, 70 eV) and scanned (m/z 35–135) at a rate of 2.5 scans/s.

To assess the potential binding of MPD and HBA, known metabolites of TBA, the LMWPF was precipitated with an equal volume of methanol and centrifuged (2000 x g, 10 min) to separate the precipitate from the supernatant. The supernatant was removed and evaporated to dryness under N2 at 55°C. The residue was reconstituted in 3-ml methanol, vortexed, and centrifuged (2000 x g, 10 min). The supernatant was removed and evaporated to dryness as described above. The residue was treated with 0.4 ml of an ethereal solution of diazomethane, for 30 min at room temperature. Derivatization of HBA to form the methyl ester was necessary due to its low volatility. The sample, containing MPD and derivatized HBA was evaporated to dryness as described above and reconstituted in 0.1-ml methanol. The sample was analyzed by GC-MS using instrumentation previously described. The inlet was operated in the splitless mode and maintained at 225°C and 2 psi (He). The column was held at 50°C for 0.1 min following injection, raised at 70°C/min to 75°C for 3.0 min, raised at 10°C/min to 175°C for 0.5 min, and finally raised at 25°C/min to 210°C for 1 min. The mass spectrometer (EI, 70 eV) was operated in the scanning mode (m/z 35–150) at a rate of 2.5 scans/s and in the SIM mode (m/z 59, 75, and 103) at a rate of 50 ms/ion. The limit of detection for MPD and HBA was estimated to be 108 pg and 359 pg (full scan mode) and 2–11 pg and 7–36 pg (SIM mode), respectively.

The LMWPF was analyzed by FPLC, and the fractions previously determined to contain 14C-TBA–derived radioactivity were pooled and concentrated as previously described. To assess the binding of TBA to {alpha}2u, an aliquot (0.2-ml) of the concentrated samples was heated at 65°C for 15 min. The headspace (0.3-ml) was analyzed by GC-MS as described above using SIM mode at m/z 59.

Protein dialysis.
Kidney cytosol, approximately 20-mg total protein, from male rats treated with 14C-TBA was dialyzed at room temperature in a Spectra/Por membrane with a molecular weight cut-off of 3500 (Spectrum Medical Industries, Inc., Houston, TX). The cytosol was dialyzed in 1.0 liter of 10-mM phosphate buffer (pH 7.2), with and without 10 g sodium dodecyl sulfate (SDS), for 16 h. Analyses were performed in triplicate. An aliquot of the dialysate (0.1-ml) was mixed with 7-ml scintillation cocktail and counted using a liquid scintillation analyzer as described above. The remaining dialysate was analyzed by gel filtration as described above.

Additional kidney cytosol (20 mg/ml) was incubated while shaking with 10 µl 0.25 M d-limonene oxide in methanol or methanol alone for 10 min at room temperature. The sample was analyzed using gel filtration, and the fractions were counted using a liquid scintillation analyzer as previously described.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Renal Concentration of {alpha}2u
The renal concentration of {alpha}2u, determined in kidney cytosol, was significantly higher (p = 0.0003) in the TBA-treated male rats (311.3 ± 56.4 µg of {alpha}2u/mg of total protein; mean ± SD) compared with corn oil-treated male rats (180.4 ± 20.1 µg of {alpha}2u/mg of total protein; mean ± SD). No histology was performed on these samples; however, results from a pilot study in which male F-344 rats were administered a single dose of 500 mg/kg TBA and euthanized 24 h following dosing, protein droplets were evident in the Mallory's Heidenhain-stained kidney sections (unpublished observation).

Measurement of TBA Equivalents in Tissue, Blood and Urine
The µmols of TBA equivalents/gram or ml of tissue or urine was greater in male rats compared to female rats in all tissues and in blood and urine (Table 1Go). Although the level in kidney was higher in male compared to female rats, it was only significant at a p value of 0.06. No difference in the percentage of the dose eliminated in urine was observed between male and female rats.


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TABLE 1 TBA Equivalents in Tissues and Urine Following a Single Dose of 500 mg/kg 14C-TBA
 
Evaluation of Chemical Binding to Kidney Protein
Analysis of kidney cytosol by gel filtration demonstrated that the high-molecular-weight protein fraction (HMWPF) and LMWPF eluted between 39 and 69 ml and 71 and 91 ml, respectively, in both male and female rats (Fig. 1Go). The {alpha}2u protein standard co-eluted with the LMWPF. Analysis of the LMWPF from 14C-TBA–treated rats demonstrated radioactivity co-eluting with the male, but not female LMWPF (Fig. 1Go). In both male and female kidney cytosol, a large portion of the radioactivity eluted as free compound (95 to 130 ml). In addition, co-elution of a small amount of radioactivity was also observed in the HMWPF in both male and female kidney cytosol (39 to 54 ml). Analysis of the LMWPF using anion-exchange chromatography demonstrated co-elution of 14C-TBA–derived radioactivity with {alpha}2u (Figs. 2A and 2BGo). The two peaks co-eluting with the {alpha}2u standard also were confirmed to be {alpha}2u, using SDS-PAGE and Western blot analysis (data not shown).



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FIG. 1. A representative analysis of kidney cytosol from 14C-TBA–treated (A) male and (B) female F-344 rats on a Sephadex G-75 gel filtration column. Both the UV spectrum (solid lines) and radiochromatogram (dashed lines) are presented. The following protein standards were used to calibrate the column (molecular weight, elution volume): blue dextran (2000 kDa, 36 ml), bovine serum albumin (67 kDa, 39 ml), ovalbumin (43 kDa, 41 ml), chymotrypsinogen A (25 kDa, 62 ml), {alpha}2u (18.7 kDa, 79 ml), and ribonuclease A (13.7 kDa, 80 ml). Abbreviations: Low-molecular-weight protein fraction (LMWPF).

 


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FIG. 2. A representative FPLC analysis of the low-molecular-weight protein fraction (LMWPF) from kidney cytosol of 14C-TBA–treated male F-344 rats. Both the UV spectrum of the LMWPF and purified {alpha}2u (A) and radiochromatogram of the LMWPF (B) are presented. The peaks of radioactivity co-elute with the {alpha}2u protein peaks in the LMWPF.

 
Analysis of TBA by GC-MS demonstrated a chromatographic retention time of 3.72 min and a mass spectrum containing a prominent m/z 59 ion (Fig. 3Go). The GC-MS analysis of the headspace from the LMWPF isolated from the kidneys of TBA-treated male rats revealed that the LMWPF contained a peak with a retention time and mass spectrum consistent with TBA (Fig. 3Go). Because acetone was identified in the urine of TBA-treated rats (Bernauer et al., 1998Go), the presence of acetone was also assessed in the headspace of the LMWPF. No acetone was detected in the LMWPF. Additional LMWPF was treated using a derivatization technique to analyze for the presence of the nonvolatile TBA metabolites MPD and HBA. Neither of these metabolites was detected in the LMWPF. The peaks containing {alpha}2u from the anion-exchange column were analyzed by GC-MS to determine if TBA was bound to {alpha}2u. TBA was not detected in these protein fractions containing {alpha}2u.



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FIG. 3. Representative GC-MS analysis of the headspace of heated kidney cytosol LMWPF from 14C-TBA–treated male F-344 rats. The (A) single ion chromatogram (m/z 59) and (B) mass spectrum for the LMWPF. Inserts represent GC-MS analysis of a TBA standard.

 
Dialysis of kidney cytosol from 14C-TBA–treated male rats was performed with d-limonene oxide, a chemical with a high affinity for {alpha}2u (Lehman-McKeeman et al., 1989Go), to determine indirectly if TBA binds to {alpha}2u. Dialysis with d-limonene oxide resulted in the disappearance of radioactivity co-eluting with the LMWPF (Fig. 4BGo). Dialysis with methanol alone did not alter the radioactivity elution pattern (Fig. 4AGo). Neither d-limonene oxide nor methanol altered the intensity of the radioactivity that co-eluted with the HMWPF.



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FIG. 4. A representative gel filtration analysis of kidney cytosol from 14C-TBA–treated male F-344 rats dialyzed with (A) methanol or (B) 0.25-M d-limonene oxide in methanol. Both the UV spectrum (solid lines) and radiochromatogram (dashed) are presented. See Figure 1Go for calibration standards.

 
Dialysis with and without SDS was used to evaluate reversible binding of TBA to {alpha}2u. 14C-TBA–derived radioactivity co-eluted with the LMWPF when kidney cytosol was dialyzed without SDS; however, radioactivity was lost from this fraction following dialysis with SDS. Following dialysis of kidney cytosol with SDS, although {alpha}2u was still present in the sample (ELISA analysis), the LMWPF peak was absent in the UV chromatogram (Fig. 5Go).



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FIG. 5. A representative gel filtration analysis of kidney cytosol from 14C-TBA–treated male F-344 rats dialyzed (A) without SDS or (B) with SDS. Both the UV spectrum (solid lines) and radiochromatogram (dashed) are presented. See Figure 1Go for calibration standards.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
From previous studies it has been shown that TBA causes protein or {alpha}2u accumulation, following inhalation exposure (Borghoff et al., 2001Go) or exposure to TBA in drinking water (Lindamood et al., 1992Go). To evaluate binding of TBA to {alpha}2u in vivo, we administered rats with TBA by gavage, which, like the other routes of exposure, was shown to increase the concentration of {alpha}2u in the male rat kidney. Our results also indicated that the µmol of TBA equivalents/gram of tissue were higher, although only slightly (p value = 0.06), in the male kidney when compared with the female kidney; 14C-TBA–derived radioactivity co-eluted with the HMWPF from male and female kidney cytosol, but only co-eluted with the LMWPF from male kidney cytosol; and 14C-TBA–derived radioactivity co-eluted with {alpha}2u from the isolated LMWPF from male kidneys, demonstrating that TBA or a metabolite of TBA interacts with or is associated with {alpha}2u in vivo. Why levels of TBA equivalents were higher in the liver and blood of male rats compared with female rats is uncertain, due to the data collection from only one time point (12 h) following dosing. GC-MS analysis confirmed that only TBA, and not MPD or HBA, metabolites of TBA, was present in the LMWPF. We were unable to confirm the presence of TBA in the isolated {alpha}2u fraction because the estimated amount of TBA in this fraction (~10 ng/ml) was below the limit of detection for the GC-MS method used. However, 14C-TBA–derived radioactivity that co-eluted with the LMWPF was collected and shown to co-elute with 2 peaks of {alpha}2u.

The binding of TBA to {alpha}2u was also assessed using a variety of equilibrium dialysis methods incorporating d-limonene oxide and SDS. Since d-limonene oxide, a known ligand for {alpha}2u, has a high affinity for {alpha}2u, d-limonene oxide would likely compete with TBA for binding to {alpha}2u (Lehman-McKeeman et al., 1989Go; Poet and Borghoff, 1997Go). We demonstrated that d-limonene oxide displaced 14C-TBA–derived radioactivity from the LMWPF that supports our hypothesis that TBA interacts with {alpha}2u. Equilibrium dialysis with SDS was used because this detergent denatures proteins, resulting in the release of bound chemical if the binding is reversible. Equilibrium dialysis of kidney cytosol from male rats treated with SDS demonstrated that the 14C-TBA was released.

Reversible binding of chemicals to {alpha}2u has been demonstrated with other inducers of {alpha}2u-N such as DCB, TMP, d-limonene, and methyl tert-butyl ether (Lock et al., 1987Go; Charbonneau et al., 1989Go; Lehman-McKeeman et al., 1989Go; Prescott-Mathews et al., 1999Go). Strong inducers of {alpha}2u such as DCB (both metabolite and parent are associated with protein) and d-limonene (parent and metabolite), as well as TMP, resulted in 12 to 40% of chemical equivalents remaining with the protein fraction dialyzed without SDS and decreasing to 0.2 to 4% with SDS. These samples were collected ~24 h after treatment. In this study only 1.5% of TBA equivalents were left after dialysis, which decreased to 0.5% with SDS. The material associated with the protein fraction after dialysis was the percentage of the total radioactivity in the kidney. This was a small amount of chemical and unlikely to be a contaminant for several reasons: First, 14C-TBA–derived radioactivity consistently co-eluted on a G-75 column with the LMWPF only in male, but not female rat kidney cytosol. Results would have been similar between male and female rats (Fig. 1Go) if TBA were contaminating the LMWPF. Second, the radioactivity in the LMWPF of male rat kidney cytosol was displaced with the addition of d-limonene-oxide, a {alpha}2u ligand, and not with methanol (Fig. 4Go), and third, 14C-TBA–derived radioactivity co-eluted with {alpha}2u separated on FPLC, and co-eluted with 2 specific {alpha}2u protein peaks (Fig. 2Go).

TBA is a mild inducer of {alpha}2u nephropathy as illustrated in Borghoff et al., 2001. It appears that only the parent, and not the metabolites of TBA are associated with {alpha}2u, unlike some of the other chemicals. The other difference between this study and some of the other studies that investigate chemical binding to {alpha}2u is the time at which the animals were killed following TBA administration (12 vs. 24 h for other studies). At an early time point, the free chemical in the kidney (vs. bound) may be higher than later time points when free chemical would be removed from the kidney and only bound chemical remain. This would result in a higher percent of the radioactivity in the kidney associated with {alpha}2u.

Also, an interesting observation was that the UV spectrum of the kidney cytosol dialyzed with SDS indicated that both the radioactivity-derived from TMP and the LMWPF peak were absent. Using an ELISA, {alpha}2u was still present in the dialyzed kidney cytosol. Other equilibrium dialysis studies with SDS, used to characterized binding of DCB, TMP, and d-limonene to {alpha}2u, did not analyze the dialyzed material for its UV elution profile. Therefore, we are unable to assess whether the change in the UV elution profile occurs with other chemicals that bind to {alpha}2u (Charbonneau et al., 1989Go; Lehman-McKeeman et al., 1989Go; Lock et al., 1987Go). Early investigations that focused on the liver synthesis of {alpha}2u suggested that covalent bonding between {alpha}2u molecules might result in dimer and trimer formation (Haars and Pitot, 1980Go). If covalent bonding between {alpha}2u molecules occurred during dialysis with SDS, then the complex would form a structure that would elute with the HMWPF. The {alpha}2u-polymers formed during this procedure and the positive ELISA could be the result of the retained epitope on this {alpha}2u complex.

TBA causes {alpha}2u accumulation and renal cell proliferation in male, but not female rats (Borghoff et al., 2001Go). The higher concentration of TBA in the male rat kidney compared with the female rat kidney, its retention, and its co-elution with {alpha}2u support that TBA interacts with {alpha}2u in vivo. Chemical binding to {alpha}2u appears critical in the accumulation of this protein (Lehman-McKeeman et al., 1990Go). When compared with TMP and other potent inducers, TBA exposure results in only mild increases in {alpha}2u concentration (Lock et al., 1987Go), suggesting that binding of TBA to {alpha}2u may not alter the rate of hydrolysis of this protein as extensively as do other {alpha}2u-inducing chemicals (Lehman-McKeeman et al., 1990Go). Alternatively, the extent of {alpha}2u accumulation could be dependent on the amount of protein with chemical bound to it, the binding affinity of the chemical to the protein, along with the degradation rate of the protein when chemically bound (Borghoff et al., 1995Go).

The U.S. EPA (1991) and the International Agency for Research on Cancer (IARC, 1998Go) developed criteria to distinguish chemicals that operate through the {alpha}2u-mediated mechanism of renal tumor formation to assess human risk associated with these chemicals. In addition to kidney tumor formation only in the male rat, these criteria include the demonstration of aspects of the pathological sequence of lesions associated with {alpha}2u nephropathy, increased number and size of protein droplets in the proximal tubule cells, renal cell proliferation, increased concentration of {alpha}2u within the protein droplets, and chemical binding to {alpha}2u. This study focused on assessing the ability of TBA to bind to {alpha}2u since other aspects of the above criteria have been previously reported (Cirvello et al., 1995Go; Borghoff et al., 2001Go; Lindamood et al., 1992Go). The demonstration of each aspect of the above criteria supports a chemical operating through the {alpha}2u-mediated mechanism and suggests that the male rat kidney tumors, associated with {alpha}2u nephropathy only, should not be used for human cancer hazard identification (U.S. EPA, 1991Go; IARC, 1998Go).


    ACKNOWLEDGMENTS
 
The authors thank the CIIT animal care staff, Horace Parkinson, Eric Howell, Erin Mooney, and Max Turner for their technical assistance, and Drs. David Dorman, Leslie Recio, Gregory Kedderis, and Barbara Kuyper for their critical review of this manuscript.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 558-1300. E-mail: borghoff{at}ciit.org. Back


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