* CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, North Carolina 277092137; and
National Institutes of Environmental Health Sciences, National Toxicology Program, Research Triangle Park, North Carolina 27709
Received June 13, 2001; accepted August 24, 2001
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ABSTRACT |
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Key Words: bromodichloromethane; BDCM; P53+/-; ; transgenic mice; C57BL/6; FVB/N; inhalation; nephrotoxicity; hepatotoxicity.
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INTRODUCTION |
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Chloroform induces liver and kidney tumors in mice and rats via a nongenotoxic-cytotoxic mode of action (Butterworth and Bogdanffy, 1999). Neither chloroform nor its metabolites are DNA-reactive mutagens. Tumor formation is secondary to initiation and promotional events associated with cytolethality, necrosis, and regenerative cell proliferation. For example, chloroform given daily by oral gavage to female B6C3F1 mice at 238 mg/kg/day induced liver necrosis and eventually liver tumors (NCI, 1976
; Larson et al., 1994a
). Similar daily doses of chloroform are achieved with 1,800,000 ppb in the drinking water. Chloroform administered to female B6C3F1 mice at 1,800,000 ppb in the drinking water induced neither liver toxicity nor liver tumors (Jorgenson et al., 1985
; Larson et al., 1994b
). Since cytotoxicity is obligatory for tumor formation, airborne concentrations of chloroform that induce organ-specific toxicity become valuable starting points for risk assessments for inhaled chloroform. Chloroform administered to male B6C3F1 mice for 13 weeks by inhalation induced regenerative cell proliferation in the liver at 90 ppm and in the kidney at concentrations of 10 ppm and above (Larson et al., 1996
). Because BDCM is similar in structure to chloroform, one purpose of these studies was to determine the shape of the dose-response curve for organ-specific toxicity for inhaled BDCM.
BDCM administered by gavage induced kidney tumors in male and liver tumors in female B6C3F1 mice at 25 and 50 mg/kg/day (Dunnick et al., 1987). BDCM given by gavage induced intestinal and kidney tumors in male F344 rats at 50 and 100 mg/kg/day and tumors in the same target organs in female F344 rats at 100 mg/kg/day (Dunnick et al., 1987
). Similar to chloroform, BDCM is less toxic when administered via drinking water compared with gavage (Lilly et al., 1994
, 1996
). Further, BDCM did not induce cancer in male B6C3F1 mice or male F-344 rats when administered in the drinking water (George and DeAngelo, 1999
). BDCM is reported to have weak genotoxic activity in some assays, but other reports describe an absence of significant genotoxic activity (Le Curieux et al., 1995
; Lipsky et al., 1993
; Morimoto and Koizumi, 1983
; Pegram et al., 1997
; Stocker et al., 1997
).
Epidemiological studies have not clearly demonstrated an association between consumption of chlorinated drinking water and increased tumor incidence in humans (Doyle et al., 1997; Cantor et al., 1998
; Flaten, 1992
; King and Marrett, 1996
). However, some studies suggest a small increase in the incidence of bladder cancer in older males consuming chlorinated drinking water (reviewed in U.S. EPA, 1998). Therefore, another purpose of the present studies was to determine whether the bladder was a target for BDCM-induced toxicity.
Many new genetically engineered mice are being proposed as tools to define mechanisms of carcinogenesis or as model systems to detect potential carcinogenic activity of chemicals using shortened exposure periods and fewer animals than in standard rodent bioassays. One such model is the p53 haploin-sufficient (p53+/-) knockout mouse. This mouse has an insert in one copy of the p53 gene rendering one allele inactive (Donehower et al., 1992); thus, only a single further mutation of the second p53 gene is required to completely inactivate this key tumor-suppressor gene. The p53+/- model shows promise for detecting potential carcinogenic activity (Tennant et al., 1995
, 1999
). This model may also have greater human relevance because the p53 gene is involved in maintaining the integrity of the DNA, is highly conserved in evolution, and is often found mutated in human cancers. Few data are available for the p53+/- model for mice exposed via inhalation or for agents that are not potent carcinogens. Ongoing validation studies utilize the p53+/- genotype on either the C57BL/6 or FVB/N genetic backgrounds. However, it is not yet known how mice representing different genetic backgrounds may interact with the p53+/--genotype, possibly resulting in variations in their toxic responses. One objective of the present study was to evaluate the role of genotype in the toxic response of the p53+/- mouse exposed to inhaled BDCM. The current study also provided the basis for selecting BDCM concentrations for the p53+/- cancer study.
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MATERIALS AND METHODS |
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Generation and characterization of atmospheres.
Mice were exposed in 1-m3 stainless steel and glass inhalation exposure chambers (Hazelton H1000, Lab Products, Seaford, DE). One 1-m3 chamber was used for each target exposure concentration. A continuous flow of HEPA-filtered air was maintained at approximately 225 l/min in each chamber. The chamber environment was maintained on a 12-h light-dark cycle at 21.7 ± 0.2°C and 50 ± 1% relative humidity. These parameters were monitored and controlled (Wong and Moss, 1996) via an automated system (Andover Controls Corp., Andover, MA).
Target exposure concentrations of BDCM were 0, 1, 10, 30, 100, or 150 ppm for the 1-week study; 0, 0.3, 1, 3, 10, or 30 ppm for the 3-week study; and 0, 0.5, 3, 10 or 15 ppm for the 13-week study. The exposure atmospheres were generated by 2 vaporization techniques. The 100 and 150 ppm concentrations were generated by metering liquid BDCM (> 98%, stabilized with K2CO3; Aldrich Chemical Company, Milwaukee, WI) into a J-tube generator. BDCM was pumped into the upper portion of the J-tube and flowed downward over glass beads which increased the surface area; nitrogen flowed up through the J-tube, which was warmed slightly to enhance vaporization. To enhance mixing, the vaporized BDCM was carried by the nitrogen stream from the top of the J-tube and introduced counter-currently into the chamber air stream approximately 6 feet (1.83 meters) upstream of the exposure chamber inlet. The 0.3- to 30-ppm exposure atmospheres were generated by metering saturated BDCM vapor from stainless steel pressure vessels of 5- to 10-gallon (18.9- to 37.9-liter) capacity. A small volume of BDCM was placed in the bottom of a pressure vessel, which was then pressurized to approximately 5 psi with nitrogen. After an overnight equilibration period, the concentrated BDCM vapor was metered through a mass flow controller (MKS Instruments, Andover, MA) and introduced countercurrently into the chamber air stream as described above.
The concentration of BDCM in the exposure chambers was monitored using a gas chromatograph (Model 5890, Hewlett Packard Co., Avondale, PA) equipped with an electron capture detector. The gas chromatograph was calibrated using certified gas standards that spanned the target concentrations up to 100 ppm. In addition, for the 1-week study, concentrations of up to 169 ppm were required and were generated by injecting liquid BDCM into Tedlar bags of known volume. For the 13-week study, a feedback control system was implemented to automatically adjust the mass flow controllers.
For the 1-week study, average concentrations in the middle dose range were 102 to 104% of the target concentration, with coefficients of variation ranging from 2.6 to 10.6%. The lowest dose average concentration was 114% of the target, with a coefficient of variation of 38.6%. The high-dose average concentration was significantly lower than the target concentration (78.8% of the target) due to problems with the metering pump system.
For the 3-week study, the averages of the daily average chamber concentrations, based on a total of 43 days of exposure, were 9297% of the targets, with coefficients of variation ranging from a low of 3.1 up to 6.9%.
For the 13-week study, the averages of the daily average chamber concentrations, based on 86 days of exposure, were 99100% of the target concentrations, with coefficients of variation ranging from 1 to 3%.
Experimental design.
Range-finding studies were performed to determine the extent of the subchronic toxicity of inhaled BDCM as well as concentrations optimal for use in a long-term cancer bioassay. Exposures were by inhalation for 6 h per day, 7 days per week, at concentrations of 0, 1, 10, 30, 100, and 150 ppm for the initial 1-week range-finding study and 0, 0.3, 1, 3, 10, and 30 ppm for the next 3-week range-finding study. Wild-type and p53+/- mice of C57BL/6 and FVB/N genotypes were analyzed for clinical and pathological changes and induced regenerative cell proliferation in target organs. The 13-week study was the first sacrifice group of a 1-year cancer study.
At 3.5 days prior to the scheduled necropsy, osmotic pumps were implanted surgically to continually administer bromodeoxyuridine (BrdU; Alzet Model 2001, Alza Corporation, Palo Alto, CA). The osmotic pumps contained a solution of 20 mg/ml filter-sterilized BrdU (Sigma Chemical Co., St. Louis MO) in phosphate-buffered saline and were implanted under aseptic conditions as described previously (Larson et al., 1994c). The mice were euthanized and necropsied for tissue collection approximately 18 hours after the final scheduled exposure.
Tissue collection and preparation.
At necropsy, mice were weighed and anesthetized with sodium pentobarbital and euthanized by exsanguination. The whole liver and both kidneys were immediately removed, weighed, and examined macroscopically. Longitudinal sections of the left and median lobes of the liver, a cross section of the right kidney, and a longitudinal section of the left kidney were fixed in 10% neutral-buffered formalin. A section of duodenum was also collected as a high-turnover tissue to verify systemic delivery of BrdU.
Bladders from all mice were collected. Sections were prepared and stained with H&E or for incorporation of BrdU.
Histopathology.
Histolopathologic changes resulting from BDCM exposure were analyzed in the kidney and liver. Changes for the kidney were scored qualitatively for severity as follows: 0 = within normal limits; 1 = minimal changes, about 10% of cortex affected; 2 = mild changes, about 25% of the cortex affected; 3 = moderate changes, about 50% of cortex affected; and 4 = severe changes, over 75% of cortex affected. Liver pathology was described as increasing in severity when centrilobular swelling, vacuolation, and degeneration, or centrilobular to midzonal vacuolation and degeneration was observed. Degenerative changes included pale eosinophilia of the cytoplasm, severe vacuolar change, and mild nuclear pyknosis.
BrdU Immunohistochemistry.
BrdU-labeled tissues were mounted on ProbeOn Plus Slides (Fisher Scientific, Pittsburgh, PA) to ensure adhesion during processing. The immunohistochemical detection of BrdU-labeled nuclei has been previously described (Eldridge et al., 1990). Briefly, sections were incubated for 1 h at room temperature with an anti-BrdU antibody (Becton-Dickinson, Mountain View, CA). After incubation with primary antibody, the slides were incubated for 30 min at room temperature with biotinylated horse antimouse IgG. Slides were then incubated with an avidin-biotin peroxidase complex (Vectastain ABC peroxidase kit, Burlingame, CA) for 30 min at room temperature. The BrdU incorporation was visualized by a final incubation with the chromagen 3-amino-9-ethylcarbazole (Zymed, San Francisco, CA) and counterstained with hematoxylin.
Scoring of labeled nuclei.
The labeling index (LI; percentage of cells in S-phase) was calculated by dividing the number of nuclei that stained positive for BrdU incorporation by the total number of nuclei counted, with the result expressed as a percentage. In the kidney, the LI was determined in the proximal tubule epithelial cells in the outer cortex as described previously (Larson et al., 1994c). In liver tissue sections, a section of the left lobe was used to determine the hepatocyte LI as described previously (Larson et al., 1994c
). A section of duodenum, representing a tissue with a relatively high cell turnover rate, was included on each slide to confirm systemic delivery of BrdU to the tissues.
The cytology/histology recognition information system (CHRIS) software package developed by Sverdrup Technology, Inc. (Sverdrup Medical/Life Sciences Imaging Systems, Fort Walton Beach, FL) was used to assess regenerative cell proliferation in the liver. Ten fields were collected in the liver, which provided approximately 1000 hepatocyte nuclei for analysis. The images were calibrated for labeled cells, unlabeled cells, and cytoplasm, and were processed as outlined in the CHRIS User's Guide provided by Sverdrup Technology, Inc. After processing, the images were reviewed for accuracy of detection and classification.
Statistics.
Comparisons of organ weights, body weights, and labeling indices were performed using an unpaired, two-tailed t-test assuming Gaussian distribution and equal variances. Significance is denoted with an asterisk (*) when p < 0.05.
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RESULTS |
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3-Week Inhalation Exposure
The 4 strains of mice were exposed for a 3-week period with atmospheric concentrations adjusted to be lower-based on results from the 1-week study. The exposure groups for the 3-week exposure were 0, 0.3, 1, 3, 10, and 30 ppm. Mortality was still observed at the 30-ppm concentration in all groups except the C57BL/6 wild-type mice (Table 1). Body weights in the treated groups were no longer significantly different from their respective controls by the 3-week time point (Table 3
). By the 3-week time point, no differences in control versus treated kidney weights were seen and the only increase in liver weights occurred at the 30-ppm dose (Table 2
). The liver and kidney lesions that were observed macroscopically and histologically after 1 week of BDCM exposure were resolving by 3 weeks, resulting in near-normal kidney architecture. Kidneys from mice exposed to the 10- and 30-ppm BDCM atmospheres had minimal to mild degenerative tubular change and regenerative tubules but did not have the acute tubular nephrosis noted at the earlier time point (Table 4
). Similarly, the high levels of regenerative cell proliferation seen in the kidney at 1 week in the 10-ppm exposure groups had returned to baseline levels by 3 weeks, with only the 30-ppm groups showing small continual elevations in LI (Fig. 3
).
The levels of hepatocyte regenerative cell proliferation seen at 1 week had returned to baseline levels by the 3-week time point (Fig. 5). The bladder was analyzed at this time point as well using standard H&E sections for pathology and BrdU immunolocalization for cell proliferation. No evidence of bladder toxicity or increases in cell proliferation was observed.
13-Week Inhalation Exposure
A 6-, 9-, and 12-month cancer bioassay was begun with the two p53+/-mouse strains, based on these range-finding studies. No wild-type mice were used for this portion of the study. A 13-week sacrifice was conducted to compare to the 1- and 3-week studies. The atmospheric concentrations chosen for these groups were 0, 0.5, 3, 10, and 15 ppm. No exposure-associated mortality or morbidity was observed at these doses for this exposure duration. At the 13-week time point, no differences in relative body, kidney, or liver weight were seen between treated and control groups (data not shown). Livers, kidneys, and bladders were examined for any signs of toxicity and changes in cell turnover. Lesions in the 13-week animals were limited to the kidneys. The C57BL/6 p53+/- animals had minimal cortical scarring and occasional regenerative tubules. Changes in FVB/N p53+/- animals were limited to mild renal cortical tubular karyocytomegaly. There was no increase over baseline in cell proliferation in the liver, kidney, or bladder.
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DISCUSSION |
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Chloroform exposure also targets the liver and kidney although the severity varied with the exposure vehicle and animal model (Butterworth and Bogdanffy, 1999). BDCM is a more potent kidney cytotoxicant than chloroform. In the mouse strains examined here, kidney lesions were evident at 10 ppm, whereas exposure concentrations of 90 ppm and 300 ppm were required in order to observe similar, chloroform-induced kidney lesions in the B6C3F1 mouse and F-344 rat, respectively (Larson et al., 1996
; Templin et al., 1996
). The more potent cytotoxicity of BDCM compared with chloroform supports a previous comparison of chloroform and BDCM in F-344 rats (Lilly et al., 1997
). This greater toxicity may be the result of differences in tissue partitioning, absorption rates, or metabolic activation (Constan et al., 1999
; Lilly et al., 1997
). Because chloroform and BDCM share a similar structure, similar enzymes likely metabolize them. Evidence suggests that BDCM, like chloroform, is activated by cytochrome P450 2E1 (Allis et al., 2001
). However, the rate of metabolism may differ such that BDCM is activated faster. Bromine is a better leaving group than chlorine. The more rapid activation in the metabolically active liver and kidney would therefore result in enhanced toxicity.
Susceptibility to BDCM's toxicity at atmospheric concentrations of 30 ppm and above varied among the backgrounds. The FVB/N background mortality rate was higher than in the C57BL/6 mice. This higher mortality appeared to be due to an increased nephrotoxcity in the FVB/N mice relative to the C57BL/6 mice. Both the observable histopathology scores and labeling index showed higher levels of toxicity at the 10- and 30-ppm doses for the FVB/N mice. No similar increase was seen in the measures of liver toxicity. In fact, liver toxicity was lower in the FVB/N relative to the C57BL/6 strain, further reinforcing kidney failure as the cause of death.
Genotypic differences in toxicity were observed as well. Differences between wild-type and p53+/- animals were observed in mortality and morbidity, body weight loss and recovery, and in the severity of liver and kidney lesions. The C57BL/6 p53+/- was more susceptible to BDCM toxicity than the wild type as measured by mortality, some histopatholgical scores, and liver labeling indices (Tables 1, 4, and 5; Figs. 3 and 5
). The same did not hold true for the FVB/N p53+/-, which was more resistant to BDCM toxicity than was the FVB/N wild type, as measured by kidney-labeling indices. Genotype-specific differences in toxic responses have been well documented in genes related to toxification and detoxification. How p53 might be linked to the activation or detoxification of BDCM is not immediately obvious. Differences in BDCM toxicity resulting from the inactivation of one p53 allele were not expected. Further, p53+/- animals have a functional p53 allele and therefore would be expected to have normal p53 function. Alterations in p53-induced gene expression have been shown in p53+/- that are distinct from the gene expression patterns observed in wild-type or p53-null animals (Gottlieb et al., 1997
; Boley et al., 2000
). Such a gene dosage effect could result from a p53 deficiency in p53+/- animals and has been termed haploinsuffiency. The results shown here indicate that the p53+/- genotype can alter the toxic response to a chemical exposure. Thus, when selecting doses for a p53+/- cancer bioassay, it would be prudent to use the p53-heterozygote rather than the wild-type surrogate.
BDCM toxicity was transient and occurred in a dose-dependent manner. Regenerative lesions characterized most, if not all, of the kidney cortex in mice exposed to high BDCM vapor concentrations for one week. After 3 weeks of exposure, damaged areas of kidney cortices entirely regenerated, only residual scarring was present, and most regenerative cell proliferation indices had returned to near baseline levels. These results are in contrast to similar experiments performed with chloroform, in which continued cytotoxicity and elevated cell turnover occurred for periods up to 90 days in both B6C3F1 mice and F-344 rats (Larson et al., 1996; Templin et al., 1996
). Why two compounds with similar structures have different effects on the targeted tissues is unclear. The ability of BDCM lesions to heal may be due to an induced change in metabolism or the emergence of a resistant cell population.
Chloroform is a nongenotoxic-cytotoxic carcinogen. Chloroform-induced tumor formation is secondary and obligatory to initiation and promotional events associated with organ-specific toxicity and regenerative cell proliferation (Butterworth and Bogdanffy, 1999). Chloroform and BDCM both induce cytolethality and cancer in the liver and kidney in various rodent bioassays. However, BDCM also induces intestinal cancer in both male and female F344 rats (Dunnick et al., 1987
).
BDCM had weak genotoxic activity in some assays, but reports are conflicting and some reports note no activity (Hayashi et al., 1988; Le Curieux et al., 1995
; Lipsky et al., 1993
; Morimoto and Koizumi, 1983
; Pegram et al., 1997
; Stocker et al., 1997
). Melnick et al. (1998) observed that BDCM induced regenerative cell proliferation in the B6C3F1 mouse liver when given by gavage. They also reported that no induced regenerative cell proliferation was seen at some lower doses where liver tumors were induced (Dunnick et al., 1987
), and therefore, concluded that cytotoxicity and cell proliferation did not play a role in BDCM-induced liver cancer. However, the only time point examined for cell proliferation was at 3 weeks (Melnick et al., 1998
). Cell proliferation was probably induced earlier, as noted in the current studies (Figs. 3 and 5
). Dramatic cell turnover, as shown in Figures 3 and 5
, in combination with modest genotoxic activity, could well initiate the cancer process, even if the duration was for less than 3 weeks.
Chlorination of drinking water produces disinfection by-products, including the trihalomethanes (THM), chloroform, and BDCM. The current U.S. EPA drinking water standard is 90 ppm for total THM. Some epidemiology reports suggest a higher incidence of bladder cancer in older men or colon or rectal cancer in people consuming chlorinated drinking water. In contrast, other studies indicate no increases in cancer. The overall weight of evidence is not sufficient to conclude an association between drinking water consumption and induced cancer in the human population (reviewed in U.S. EPA, 1998). As part of these studies, bladders were examined for induced pathological changes or regenerative cell proliferation, and none were observed. Thus, the bladder does not appear to be a target organ for BDCM in the strains of mice examined here.
One purpose of the current study was to establish appropriate doses for a one-year p53+/- cancer bioassay with inhaled BDCM. The current profiles of toxicity provide valuable data regarding the early events following BDCM exposure. This new data allows comparisons to be made between early organ-specific toxicity and any cancers induced by this potent kidney and liver toxicant.
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ACKNOWLEDGMENTS |
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NOTES |
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