CIIT Centers for Health Research, PO Box 12137, Six Davis Drive, Research Triangle Park, North Carolina 27709-2137
Received November 29, 2000; accepted January 8, 2001
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ABSTRACT |
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Key Words: tert-butyl alcohol (TBA); 2u-globulin (
2u)..
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INTRODUCTION |
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TBA is a metabolite of MTBE in both rodents and humans. The pharmacokinetics of TBA following MTBE exposure are similar in these species; TBA accumulates in blood and is slowly cleared from blood (Amberg et al., 1999; Miller et al., 1997
; Nihlen et al., 1998
). In 13C-TBAdosed rats, 13C-acetone, TBA, and its glucuronide were found to be minor metabolites and 2-methyl-1,2-propanediol and 2-hydroxyisobutyrate were the major metabolites present in urine, demonstrating that TBA is oxidatively metabolized or directly conjugated in vivo (Bernauer et al., 1998
).
In a 2-year cancer study, administration of various levels of TBA in drinking water significantly increased the incidence of renal tumors in male F-344 rats and follicular cell adenomas of the thyroid in female mice (Cirvello et al., 1995; NTP, 1995
). TBA did not induce any mutations when tested in four different strains of Salmonella typhimurium (Zeiger et al., 1987
). More recently, TBA was shown to cause an increase in mutations in the TA102 strain of Salmonella following 24 mg of TBA/plate (Williams-Hill et al., 1999
), but was negative in L5178Y mouse lymphoma cell assay with and without metabolic activation (McGregor et al., 1988
). Overall, TBA is considered to cause tumors in rodents through a nongenotoxic mechanism.
A number of nongenotoxic chemicals that cause male rat-specific renal tumors also induce a syndrome unique to male rats, referred to as protein droplet, or 2u-globulin (
2u), nephropathy (
2u-N) (U.S. EPA, 1991b
). More recently, MTBE has been added to this list (Bird et al., 1997
; Prescott-Mathews et al., 1999
). The proposed mechanism for
2u-N is dependent on the accumulation of the male ratspecific, low-molecular-weight protein
2u within the renal proximal tubule. Following glomerular filtration of
2u, approximately 50% of the protein is reabsorbed by proximal tubule cells, primarily in the P2 segment, while the remainder is excreted in the urine (Neuhaus et al., 1981
). Under normal conditions, reabsorbed
2u is degraded by proteolytic enzymes within the phagolysosomes. Following exposure to
2u inducers, however, the parent compound or a metabolite binds
2u in a noncovalent, reversible manner, rendering
2u resistant to proteolytic hydrolysis. Massive accumulation of
2u overwhelms the phagolysosome, resulting in proximal tubule necrosis and compensatory cell proliferation (see review by Swenberg and Lehman-McKeeman, 1999). A sustained increase in cell proliferation is capable of increasing the likelihood of converting a spontaneous or chemically mediated error in DNA replication to a mutation and may enhance the clonal expansion of the initiated cell population to an eventual tumorigenic response (Butterworth et al., 1992
).
2u-N is a male ratspecific response and does not occur in female rats or other species, including mice, guinea pigs, hamsters, dogs, and nonhuman primates (reviewed by Borghoff et al., 1990). Additionally, no evidence to date supports the synthesis of
2u or the development of
2u-N in humans (Borghoff and Lagarde, 1993
; Lehman McKeeman and Caudill 1992
; Olson et al., 1990
).
In a 90-day TBA drinking water study, direct effects of TBA were found only in the kidney, ureter, and urinary bladder of exposed F-344 rats (Lindamood et al., 1992; Takahashi et al., 1993
). Hyaline (protein) droplet deposition and hyaline crystals in tubular epithelium were enhanced in all male rats exposed to TBA, with the exception of the high-dose group (4% TBA). Enhanced cell proliferation, indicated by an increase in proliferating cell nuclear antigen (PCNA)positive S-phase cells, was evident in the male rats exposed to 1% and 2% TBA. These changes correlated with the deposition of protein in the kidneys at the same concentrations (Lindamood et al., 1992
; Takahashi et al., 1993
). Additionally, histopathologic examination of hematoxylin-eosin (H&E)-stained male rat kidney sections revealed a dose-related increase in the severity of renal lesions, characterized by tubular atrophy, tubular regeneration, or thickening of the basement membranes in male rats administered 0.25, 0.5, 1.0, and 2.0% TBA, but not 4% TBA.
Although the TBA drinking water studies indicate that TBA induces protein droplet nephropathy in male F-344 rats, no investigation thus far has definitively demonstrated an exposure-related accumulation of 2u or specific renal cell proliferation in the male rats. Therefore, the objective of the present study was to determine whether TBA induces
2u-N and enhanced renal cell proliferation in male, but not female, F-344 rats, and if the dosimetry of TBA to the kidney is gender specific. Because water consumption was decreased for a considerable period of time in rats exposed to TBA in their water compared to controls (Lindamood et al., 1992
), we elected to expose rats to TBA via inhalation. Based on review of archival H&E-stained kidney sections from an 18-day TBA inhalation study, exposure concentrations of 0, 250, 450, and 1750 ppm TBA were selected for the present investigation (NTP, 1997
). To evaluate sex-specific retention of TBA, the disposition of TBA to the kidney of male and female rats exposed to TBA either for 1 day or 8 days via inhalation was also carried out. As
2u-N is a syndrome unique to male rats, elucidation of the mechanism of TBA-induced renal carcinogenesis is important not only in assessing the relevance of this response to humans, but also in understanding the role of TBA in the MTBE-induced renal tumor response in male rats.
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MATERIALS AND METHODS |
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Animal exposures.
Male and female rats were randomized (n = five rats/sex/concentration) prior to exposure. Animals were housed and exposed to TBA vapors for 6 h/day in 1-m3 H1000 stainless steel and glass chambers (H1000, Lab Products, Inc., Maywood, NJ). The H1000 chambers were contained within permanent 8-m3 Hinners-type chambers. The 8-m3 chambers served as an additional safety containment system. Temperature was maintained between 2026°C and relative humidity between 4060%. Air flow through the chamber ranged from 200 to 250 l/min, which maintained 1215 air changes/h.
The exposure atmospheres of TBA (> 99% pure, CAS No. 75-65-0, Mallinckrodt, Raleigh, NC) were generated by metering known amounts of TBA with a fluid metering pump (Fluid Metering Inc., Oyster Bay, NY) into a stainless steel J-tube. The reservoir containing the TBA was immersed in a water bath maintained between 34 and 38°C to prevent the TBA from solidifying. Nitrogen was passed through the J-tube to carry the TBA vapor into the air supply of a 1-m3 exposure chamber. Prior to the start of exposures, uniformity of distribution of TBA in each exposure chamber was checked by measuring the concentration at nine positions (eight corners and the center of the chamber). The variation of concentration within the chamber was < 2% for all chambers.
The concentration of the test atmosphere in each chamber was analyzed approximately three time/h using a gas chromatograph (GC) (Hewlett Packard 5890 series II) equipped with a multiport sampling valve, a 0.5-ml sample loop, a flame ionization detector, and a 5-ft x 1/8" OD stainless steel column packed with 1.5% OV-101 on Chromasorb G-HP (Alltech, Deerfield, IL). Target concentrations of 0, 250, 450, and 1750 ppm TBA were selected based on results from a 18-day vapor inhalation study conducted by the National Toxicology Program (NTP, 1997). The GC was calibrated by preparing sample bags of known TBA concentrations.
Study design to evaluate TBA-induced 2u-nephropathy.
A total of 80 rats (40 males and 40 females) was used in the experiment to assess 2u-globulin nephropathy (
2u-N). Male and female rats were assigned to one of two subgroups (five rats/sex/exposure concentration/subgroup) for determination of either histologic or biochemical end points and exposed to target concentrations of 0, 250, 450, or 1750 ppm TBA for 6 h/day for 10 consecutive days.
Tissue preparation for histopathology end points.
At 3.5 days prior to euthanasia, five male and five female rats from each exposure group were subcutaneously implanted with an osmotic pump (Alzet model 2ML1, Alza Corporation, Palo Alto, CA) for delivery of 5-bromo-2-deoxyuridine (BrdU). One day following the final exposure, rats were anesthetized with sodium pentobarbital, and the kidneys were perfused with 2% paraformaldehyde/1% glutaraldehyde via a retrograde intravascular perfusion technique through the descending aorta, as previously described (Larson et al., 1993). A midlongitudinal section of the left kidney, midtransverse section of the right kidney, and transverse sections of the duodenum were fixed in a 2% solution of paraformaldehyde and 1% glutaraldehyde. The fixative was replaced after 24 h with 70% ethanol. Tissues were embedded in paraffin and serial sections cut at 5 µm. Tissue sections were stained with H&E and Mallory's Heidenhain stain (Cason, 1950
) to evaluate histologic lesions and protein droplet accumulation, respectively. Histologic end points evaluated were renal lesions, protein droplet accumulation, quantification of the labeling index (LI) by immunohistochemical staining for BrdU, and determination of
2u accumulation within renal tubules by
2u immunohistochemical staining.
Histologic evaluation.
Slides stained with Mallory's Heidenhain, a nonspecific protein stain (Cason, 1950), were evaluated according to the relative accumulation or severity of the protein droplets. Protein droplet accumulation was graded on a relative scale of 04, based on the size of protein droplets and intensity of staining, with no prior knowledge of treatment group. Scores were assigned to kidney sections from each animal as follows: 0, no protein droplets observed; 1, occasional small protein droplets observed; 2, small protein droplets frequently observed; 3, small to moderately sized protein droplets frequently observed; and 4, large protein droplets seen within many proximal tubular epithelial cells. Lesions were also graded on a scale of 04 with no knowledge of treatment group according to the percentage of affected renal tubules throughout the cortex and outer stripe of the outer medulla (OSOM). Scores were assigned to kidney sections from each animal as follows: 0, absence of lesions; 1, < 10% of the tubules affected; 2, 1125% of the cortex and OSOM involved; 3, 2650% of the tubules affected; and 4, > 50% of the cortex and OSOM involved. The two individual scores were multiplied to yield a grading scale of 016 (Lehman-McKeeman and Caudill, 1999
).
Cell proliferation.
Immunohistochemical staining for BrdU incorporation was performed on male and female rat kidney and duodenum using a streptavidin alkaline phosphatase technique and Stable Fast Red chromagen. A mouse monoclonal antibody raised toward purified BrdU (Caltag, South San Francisco, CA) and biotin conjugated antimouse IgG (Vector Laboratories, Inc., Burlingame, CA) were used as the primary and secondary antibodies, respectively. A section of duodenum, which normally exhibits a high cell proliferation rate, was included to confirm systematic delivery of BrdU. Tissue sections were processed for BrdU immunohistochemistry according to a modified method of Eldridge et al. (1990) described by Prescott-Mathews et al. (1999). Cell proliferative responses were evaluated through determination of the LI, as described by Prescott-Mathews et al. (1999). The LI was defined as the percentage of positively stained epithelial cells within the proximal tubule. Labeled cells throughout the renal cortex were counted. A total of 2000 tubular epithelial cells were counted within the renal cortex at 400x magnification. In the left kidney, 1000 cells were counted by beginning at the outermost portion of the cortex and counting diagonally inward toward the medulla. Quantification of 1000 cells in the right kidney was accomplished by counting diagonally from the innermost portion of the renal cortex and counting outward.
2u immunohistochemical staining.
Immunohistochemical staining of 2u was performed on male and female rat kidney sections using a streptavidin horseradish peroxidase technique and aminoethyl carbazole chromagen. A mouse monoclonal antibody (prepared by Hazelton Biotechnologies Co., Vienna, VA) raised toward purified rat urinary
2u (Kurtz et al., 1976
) was used as the primary antibody. The mouse monoclonal antibody did not demonstrate any crossreactivity with other proteins in kidney cytosol using SDS-PAGE and Western blot analysis (unpublished observation).
2u immunohistochemical staining was performed as previously described by Burnett et al. (1989), with modifications described by Prescott-Mathews et al. (1999).
Tissue preparation for biochemical end points.
Five male and five female rats from each exposure group were euthanized by CO2 asphyxiation. Animals were exsanguinated via the caudal vena cava. Kidneys were excised with the capsules removed, weighed, and homogenized in ice-cold 3x (w/v) 0.1 M Na/K phosphate buffer, pH 7.4. Kidney homogenate was stored at 70°C. Prior to measurement of 2u concentration by the enzyme-linked immunosorbent assay (ELISA), the kidney homogenate was thawed on ice, and kidney cytosol was prepared by centrifugation at 600 x g for 15 min at 4°C. The resultant supernatant was then centrifuged at 116,000 x g for 60 min at 4°C. Aliquots of the supernatant were then stored at 70°C.
Enzyme-linked immunosorbent assay.
The renal concentration of 2u was measured in male and female rat kidney cytosol using an ELISA with a mouse monoclonal antibody directed against purified rat urinary
2u (prepared by Hazelton Biotechnologies Co., Vienna, VA). Kidney cytosol prepared from male rats treated for 4 days with 500 mg/kg 2,2,4-trimethylpentane (TMP) (Prescott-Mathews et al., 1997) was used as a positive control on each plate of samples analyzed. Urinary
2u was purified according to a modified method of Kurtz and coworkers (1976). Using this ELISA technique,
2u was quantified using anti-mouse IgG alkaline phosphatase conjugate and p-nitrophenyl phosphate, according to a previously described method (Borghoff et al., 1992
). The
2u concentration in the kidney cytosol was expressed as micrograms
2u per milligram total protein. Total protein was measured using the BCA protein assay (Pierce, Rockford, IL), with BSA as the standard.
Study design to evaluate the dosimetry of TBA.
The dosimetry of TBA was assessed in male and female rats following single and repeated (8-day) exposures to target concentrations of 250, 450, or 1750 ppm TBA (n = three rats/sex/exposure concentration/time point) for 6 h/day. Rats were killed at various time points following exposure (2, 4, 6, 8, and 16 h) to measure levels of TBA in the liver, kidney, and blood. These animals were housed in the same chambers as the rats evaluated for 2u-N. The earliest time point of 2 h following exposure was selected based on coordination of removal of animals from chambers, along with the knowledge that TBA is cleared slowly from blood (half-life
5 h). Although elimination within the first 2 h following the cessation of exposure is critical for pharmacokinetic evaluation, the objective of this study was to compare tissue levels of TBA in male and female rats following single and repeated exposure to TBA. Eight days of repeated exposure was selected based on our ability to handle animals designated to evaluate the various end points described above.
Analysis of tissue TBA levels.
Following exposure, rats were exposed to CO2 ( 11.5 min) at various time points, and blood was withdrawn from the vena cava. Three 1-ml samples were added directly to 10-ml headspace vials and sealed immediately. Three 1-g liver samples and each kidney were minced in 2 ml 0.1 M succinic acid buffer, pH 4.5, in 10-ml headspace vials, and the vial was sealed. Tissue samples were heated at 60°C for 30 min prior to analysis. A 1-ml aliquot of the headspace from each tissue sample was analyzed for TBA using a Headspace Autosampler Model 7694 (Hewlett Packard, Avondale, PA) linked to a Hewlett Packard 5890 Series II gas chromatograph fitted with a capillary injection port, a flame ionization detector, and a Hewlett Packard 3396 Series Integrator. TBA was separated using a series of columns consisting of a capillary SPB-1 precolumn (11 x 0.53 mm; Supelco, Bellefonte Park, PA) connected to a capillary DB-Wax column (30 x 0.53 mm; Alltech, Deerfield, MA) attached to a capillary SPB-5 column (30 x 0.53 mm; Supelco, Bellefonte Park, PA). The oven temperature was initially held at 45°C for 9 min, increased at 70°C/min to 175°C, and held for 4 min. The injection and flame ionization detector temperatures were 200°C and 250°C, respectively. Helium was used as a carrier gas at a flow rate of approximately 4 ml/min at a pressure of 7.3 psi. Individual tissue standard curves were generated by incubating known amounts of TBA with tissue samples obtained from untreated rats under the identical conditions to the unknown samples. The TBA tissue:blood ratio was calculated by dividing the level of TBA in the tissue (liver or kidney) by the level in blood of each rat.
Statistical analysis.
Body and tissue weight comparisons were made using Student's t-test and Tukey-Kramer one-way analysis of variance (ANOVA) with a level of significance of p < 0.05. LI, ELISA, and protein droplet accumulation data were analyzed by Tukey-Kramer one-way ANOVA with a level of significance of p < 0.05. An arcsine transformation was applied to the LI data to meet criteria for normality and homogeneity for ANOVA. The square root of the protein droplet accumulation score (016) was used so that the assumption of normality was met. Dunnett's test (p < 0.05) was also carried out to assess the accumulation of protein droplets with exposure concentration.
To analyze trends in protein droplet accumulation and LI with exposure concentration and the correlation between 2u levels and LI, linear regression analysis was used. Trends were found to be significant, with a level of p < 0.05.
Tissue:blood ratios were compared using one-way ANOVA with a level of significance of p < 0.05.
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RESULTS |
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DISCUSSION |
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Although 2u immunoreactivity was present in protein droplets in kidneys from TBA-exposed male rats, a linear, concentration-related increase in
2u staining was not observed. Similar findings were observed in the MTBE 10-day and 13-week inhalation studies in which grading of
2u-stained kidney sections did not reveal any linear concentration-related increase in
2u staining in MTBE-exposed male rats (Prescott-Mathews et al., 1999; J. A. Swenberg and D. R. Dietrich, personal communication). In the 10-day study, Prescott-Mathews et al. (1999) observed that the concentration of
2u increased in kidney cytosol using an ELISA. The same assay was used to show an increase in
2u concentration in the kidneys of male rats exposed to 1750 ppm TBA for 10 days. A strong
2u inducer such as TMP caused a greater increase in
2u accumulation than what was shown for either MTBE or TBA when measured by the ELISA (Prescott-Mathews et al., 1999
). Staining of protein droplets for
2u and the level of protein droplet accumulation were also more intense with this chemical (Short et al., 1989
). The mild increase in
2u concentration in TBA-exposed male rats may only be detectable by quantitating the level of
2u using an ELISA, and not by evaluating immunohistochemical staining for
2u, as the former is a more sensitive method.
Characteristic renal lesions consistent with 2u-N, such as accumulation of granular casts and linear mineralization of the kidney, are typically observed in studies with continued exposure for 2890 days. Data from a 90-day drinking water study confirm TBA-induced protein droplet accumulation within the proximal tubule (Lindamood et al., 1992
). Effects included increased severity of lesions characteristic of chronic progressive nephropathy (greater in female rats) and increased incidence and severity of mineralization (greater in male rats). Both nephropathy and mineralization were reduced at the highest dose level of 4% TBA (3588.5 mg/kg/day) (Table 3
) compared with the 2% TBA level (1598.9 mg/kg/day), which may have been influenced by the high mortality at 4% level. Protein droplet exacerbation was increased in male rats up to 2% TBA, along with an increase in PCNA-stained S-phase nuclei. Both responses decreased in the rats exposed to 4% TBA in their water, compared with the group exposed to 2% TBA.
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The current hypothesis for 2u-mediated renal tumorigenesis relies on the accumulation of a chemical
2u complex resistant to proteolytic degradation in the phagolysosome. As such,
2u accumulates in and appears to overwhelm the phagolysosome, resulting in cell death and regenerative cell proliferation (Borghoff et al., 1990
). Enhanced renal cell proliferation, as determined by the LI, was observed in male rats exposed to TBA. Whether
2u accumulation was totally responsible for the increase in cell proliferation observed only in male rat kidney following exposure to TBA is not clear from this investigation. In Table 3
, doses were estimated from the various TBA exposure studies and responses compared. In the 90-day drinking water study and this 10-day inhalation study, similar doses (albeit via different routes of administration) apparently induce protein droplet accumulation. In the 2-year chronic study, however, the dose of TBA that caused renal tumors was lower than the dose necessary to cause protein droplets, which may suggest a possible second mechanism of toxicity involved in the tumor formation.
Even though the increase in 2u concentration correlated with an increase in cell proliferation in this study, when examining the response at the lowest exposure concentration (250 ppm), there was no significant increase in the level of
2u or the accumulation of protein droplets, whereas the LI was significantly increased. It is possible that measuring the LI in the kidney using a 3-day BrdU pump is a more sensitive method than evaluating protein accumulation or measuring an increase in the concentration of
2u in the kidney at one time point following exposure. With strong inducers, there is information to demonstrate that the maximum difference in
2u concentration between controls and treated rats is approximately 24 h following chemical treatment. However, the time course for an increase in
2u concentration in the kidney following mild inducers may be different. The difference in sensitivity of the assays used for measuring
2u and the LI, the time course of these responses, or the potential of a second mechanism of toxicity are possible explanations for the lack of correlation between
2u and the LI at low TBA exposure concentrations.
TBA-induced 2u-N may be necessary in the development of renal tumors in male rats, but not sufficient. Other factors in addition to
2u-N that may contribute to the renal cell proliferative response in male rat kidneys following TBA exposure were not obvious from this study. TBA is metabolized to 2-hydroxyisobutyrate and 1,2-methyl-2-propanediol. These metabolites are eliminated in the urine of rats and humans exposed to MTBE or TBA (Amberg et al., 1999
; Bernauer et al., 1998
; Miller et al., 1997
). The presence of these compounds in urine provides evidence that TBA is oxidized. In this investigation, we have shown that TBA appears to accumulate in the male rat kidney with repeated TBA exposure. TBA was also cleared more slowly from the male rat kidney compared with the female rat kidney. Levels of TBA were detected in the male rat kidney 16 h following exposure. Binding of TBA to
2u would explain this male ratspecific retention of TBA. It is also important to note that the male and female rat kidney were exposed to similar concentrations of TBA; however, an increase in cell proliferation was only observed in the male rat kidney (Fig. 9
). This demonstrates that TBA itself is not directly responsible for the enhanced cell proliferation that occurs in the male rat kidney.
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ACKNOWLEDGMENTS |
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NOTES |
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