CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, North Carolina 27709
Received December 29, 2000; accepted May 7, 2001
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ABSTRACT |
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Key Words: tert-butyl alcohol (TBA); 2u-globulin (
2u) nephropathy; male rats; protein binding.
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INTRODUCTION |
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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., 1995; NTP, 1995
). Although one recent study demonstrated that TBA was mutagenic in the Ames assay using the TA102 strain (Williams-Hill et al., 1999
), in all other systems tested, TBA was found to be negative (McGregor et al., 1988
; NTP, 1997
; Zeiger et al., 1987
). 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., 1992; Takahashi et al., 1993
). These features are characteristic of
2u-globulin nephropathy (
2u-N), a syndrome considered unique to male rats, which is characterized by the accumulation of
2u-globulin (
2u), a low-molecular-weight protein, in the form of protein droplets within the renal proximal tubule (Borghoff et al., 1990
; Swenberg and Lehman-McKeeman, 1999
). In this study analyses were not performed to definitively confirm the presence of
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
2u accumulation.
To further investigate the ability of TBA to cause 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
2u (Borghoff et al., 2001
). TBA also increased the concentration of
2u and enhanced cell proliferation in only male rat kidneys. The increase in
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
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., 1989
), TMP (Lock et al., 1987
), or d-limonene (Lehman-Mckeeman et al., 1989
). 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 2u-N, either the parent compound and/or a metabolite binds to
2u in a reversible, noncovalent manner. The formation of this chemical-
2u complex renders the protein resistant to hydrolysis (Lehman-McKeeman et al., 1990
). As such,
2u accumulates in proximal tubule epithelial cells leading to necrosis and enhanced renal cell proliferation (Borghoff et al., 1990
; Hard et al., 1993
; Swenberg and Lehman-McKeeman, 1999
).
Since TBA administration (either orally or via inhalation) to male rats causes 2u accumulation in the kidney, we hypothesized that TBA, or its metabolites, interacts with
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
-hydroxyisobutyric acid (HBA), bind to
2u. Since chemical binding to
2u and the formation of a chemical-
2u complex is critical to the development of
2u-globulin nephropathy, demonstration of binding would provide additional information to support the classification of TBA as an inducer of
2u-globulin nephropathy. If the TBA-induced kidney tumors in male rats is mediated through the ability of TBA to induce
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, 1991
). These data are critical for human health risk assessment for this chemical.
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MATERIALS AND METHODS |
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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 1214 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 3070%. 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, 1995
). 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
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-TBAtreated 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 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 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 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
2u, as described by Borghoff and Lagarde (1993).
Measurement of 2u.
The concentration of 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
2u was used (prepared by Hazelton Biotechnologies Co., Vienna, VA). The monoclonal antibody directed against
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
2u-globulin nephropathy, was used as a positive control (Prescott-Mathews et al., 1997
).
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 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-TBAtreated 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-TBAderived 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-2u antibody as described by Borghoff and Lagarde (1993) to determine whether the 14C-TBAderived radioactivity co-eluted with
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 35135) 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 35150) 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 211 pg and 736 pg (SIM mode), respectively.
The LMWPF was analyzed by FPLC, and the fractions previously determined to contain 14C-TBAderived radioactivity were pooled and concentrated as previously described. To assess the binding of TBA to 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.
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RESULTS |
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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 1). 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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DISCUSSION |
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The binding of TBA to 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
2u, has a high affinity for
2u, d-limonene oxide would likely compete with TBA for binding to
2u (Lehman-McKeeman et al., 1989
; Poet and Borghoff, 1997
). We demonstrated that d-limonene oxide displaced 14C-TBAderived radioactivity from the LMWPF that supports our hypothesis that TBA interacts with
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 2u has been demonstrated with other inducers of
2u-N such as DCB, TMP, d-limonene, and methyl tert-butyl ether (Lock et al., 1987
; Charbonneau et al., 1989
; Lehman-McKeeman et al., 1989
; Prescott-Mathews et al., 1999
). Strong inducers of
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-TBAderived 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. 1
) 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
2u ligand, and not with methanol (Fig. 4
), and third, 14C-TBAderived radioactivity co-eluted with
2u separated on FPLC, and co-eluted with 2 specific
2u protein peaks (Fig. 2
).
TBA is a mild inducer of 2u nephropathy as illustrated in Borghoff et al., 2001. It appears that only the parent, and not the metabolites of TBA are associated with
2u, unlike some of the other chemicals. The other difference between this study and some of the other studies that investigate chemical binding to
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
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, 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
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
2u (Charbonneau et al., 1989
; Lehman-McKeeman et al., 1989
; Lock et al., 1987
). Early investigations that focused on the liver synthesis of
2u suggested that covalent bonding between
2u molecules might result in dimer and trimer formation (Haars and Pitot, 1980
). If covalent bonding between
2u molecules occurred during dialysis with SDS, then the complex would form a structure that would elute with the HMWPF. The
2u-polymers formed during this procedure and the positive ELISA could be the result of the retained epitope on this
2u complex.
TBA causes 2u accumulation and renal cell proliferation in male, but not female rats (Borghoff et al., 2001
). The higher concentration of TBA in the male rat kidney compared with the female rat kidney, its retention, and its co-elution with
2u support that TBA interacts with
2u in vivo. Chemical binding to
2u appears critical in the accumulation of this protein (Lehman-McKeeman et al., 1990
). When compared with TMP and other potent inducers, TBA exposure results in only mild increases in
2u concentration (Lock et al., 1987
), suggesting that binding of TBA to
2u may not alter the rate of hydrolysis of this protein as extensively as do other
2u-inducing chemicals (Lehman-McKeeman et al., 1990
). Alternatively, the extent of
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., 1995
).
The U.S. EPA (1991) and the International Agency for Research on Cancer (IARC, 1998) developed criteria to distinguish chemicals that operate through the
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
2u nephropathy, increased number and size of protein droplets in the proximal tubule cells, renal cell proliferation, increased concentration of
2u within the protein droplets, and chemical binding to
2u. This study focused on assessing the ability of TBA to bind to
2u since other aspects of the above criteria have been previously reported (Cirvello et al., 1995
; Borghoff et al., 2001
; Lindamood et al., 1992
). The demonstration of each aspect of the above criteria supports a chemical operating through the
2u-mediated mechanism and suggests that the male rat kidney tumors, associated with
2u nephropathy only, should not be used for human cancer hazard identification (U.S. EPA, 1991
; IARC, 1998
).
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ACKNOWLEDGMENTS |
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NOTES |
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