BASF Aktiengesellschaft, Product Safety, Z 470D-67056 Ludwigshafen, Germany
Received April 23, 2002; accepted July 1, 2002
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ABSTRACT |
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Key Words: tetrahydrofuran; butylene oxide; rat; mouse; liver; kidney; 2u-globulin; tumor; cell proliferation; apoptosis.
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INTRODUCTION |
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The mutagenic activity of THF was investigated in a variety of in vitro and in vivo assays. It was not mutagenic in Salmonella typhimurium, and did not induce sister chromatid exchanges or chromosomal aberrations in cultured Chinese hamster ovary cells (CHO). No increase in sex-linked recessive lethal mutations was detected in germ cells of male D. melanogaster exposed to THF. Results of in vivo assays for induction of chromosomal aberrations, sister chromatid exchanges in mouse bone marrow, and a mouse micronucleus test were negative (Matthews et al., 1996; Mortelmans et al., 1996; U.S. Department of Health and Human Services, 1998).
However, the results of chronic inhalation studies in F344/N rats and B6C3F1 mice exposed to 0, 200, 600, or 1800 ppm (0, 600, 1800, and 5400 mg/m3) THF, 6 h per day, 5 days per weeks, for 105 weeks were positive for carcinogenicity as follows: THF caused an increase in the incidence of epithelial renal tubule adenoma or carcinoma in male rats at 600 and 1800 ppm (the combined incidences were 1/50, 1/50, 4/50, and 5/50 at 0, 200, 600, and 1800 ppm, respectively) and an increase in the incidence of liver adenoma or carcinoma in female mice at 1800 ppm (the combined incidences were 17/50, 24/50, 26/50 and 41/48 at 0, 200, 600, and 1800 ppm, respectively) (U.S. Department of Health and Human Services, 1998).
The purpose of the present study was to investigate the possible non-genotoxic mode of action leading to an increase in liver tumors. Such increased incidence, particularly in sensitive strains such as the B6C3F1 mouse, is often associated with enzyme induction and cell proliferation. Therefore, the following parameters were investigated in the mouse studies: enzyme induction, cell proliferation and apoptosis, in female mouse liver
Increased incidence of kidney tumor formation in male rats with non genotoxic compounds has been frequently associated with the accumulation of 2u-globulin (
2u). The mode of action considered responsible for the carcinogenic effect on the kidney involves a sustained regenerative cell proliferation throughout the duration of exposure (Hard, 1998
) due to indirect cytotoxicity associated with lysosomal overload caused by binding to
2u-globulin in male rats.
Therefore, the following parameters were investigated in the rat studies: enzyme induction, cell proliferation, specific immunohistochemistry indicative of 2u accumulation, and apoptosis in male rat kidney.
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MATERIALS AND METHODS |
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Animals and maintenance conditions.
Male Fischer 344 rats (CDF [F-344]/CRL BR) were supplied by Charles River Deutschland, Sulzfeld; Germany. Female B6C3F1 mice (Rj IOPS) were supplied by Centre Elevage R. Janvier, Le Genest St. Isle, France. The age of the animals was approximately 10 weeks at delivery. The animals were singly housed in wire cages (type DK III for rats, floor area 800 cm2; type DK I for mice, floor area 200 cm2) supplied by Becker & Co., Castrop-Rauxel, Germany. Waste trays were fixed underneath the cages, containing bedding material (type dust free, supplied by SSNIFF, Soest, Germany) for rats, and paper for mice. The animals were maintained in an air-conditioned room at a temperature of 2024°C, a relative humidity of 3070%, and a 12-h light/12-h dark cycle. Before the animals arrival, the room was completely disinfected with AUTEX, a fully automatic, formalin ammonia-based terminal disinfectant supplied by Dr. Gruss KG, Neuss, Germany). During the study, the floor and walls were cleaned weekly with a solution of 1% Mikroquat® in water. The animals were maintained on rat/mouse/hamster laboratory diet, 10-mm pellets (Provimi Kliba SA, Kaiseraugst, Switzerland) and tap water ad libitum. Food and drinking water was assayed for chemical as well as for microbiological contaminants.
Experimental design.
The main study (Table 1) was comprised of 4 groups of 6 male rats and 10 female mice that were exposed to dynamic atmospheres containing THF concentrations of 0, 600, 1800, or 5400 mg/m3 for 6 h per day, 5 days per week, for 4 weeks (20 daily exposures in total); the animals were sacrificed the day after the last exposure. In 2 satellite studies, groups of male rats and female mice received the same THF treatment regimen for only 5 days. In the first satellite study, the animals were sacrificed immediately after their last exposure. In the second satellite study, the animals were sacrificed 21 days after the last exposure. The body weights of male rats and female mice at the start of the THF exposure period ranged from 234 to 248 g and from 22.0 to 23.2 g, respectively.
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Analytical determination of THF concentrations.
The THF concentrations in the inhalation atmospheres were determined daily by gas chromatography (Hewlett-Packard 5840A) in each of 2 samples from the breathing zone of the animals per THF treatment group. In the control group, weekly samples were analyzed using xylene as solvent, on a weekly basis. Additionally, the constancy of THF concentrations in the inhalation atmospheres throughout each exposure was monitored continuously by total hydrocarbon analyzers (Fidamat Siemens) for 600 mg/m3 and Testa 123 for 1800 and 5400 mg/m3), recorded using line recorders, and transferred to the automated measuring system.
Clinical observations.
The general state of health of the animals was checked twice daily on working days and once daily during weekends or on holidays. On THF exposure days, clinical examinations were performed before, during, and after exposure. Body weights were determined at the start of the pre-flow period, at the start of the THF exposure period, and at weekly intervals, thereafter.
Preparation of S9 fraction and microsomes from liver and kidney.
The organs were cut into pieces and homogenized in 250 mM sucrose/1 mM Na-EDTA. The homogenate was centrifuged at 9000 x g for 15 min at 4°C. Part of the supernatant was used as the S9 fraction. The remaining supernatant was then centrifuged at 100,000 x g for 60 min at 4°C. Microsomal pellets were recovered, resuspended by homogenization, and again centrifuged at 100,000 x g. Microsomal pellets were recovered and stored at 80°C until use.
Microsomal enzyme determinations.
Cytochrome P450 content and ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-depentylase (PROD) activities were determined in the kidneys of 5 male rats and the livers of 5 female mice exposed to 0 or 5400 mg/m3 for 5 consecutive days (i.e., control and high-dose animals from the first satellite study). The cytochrome P450 content was measured photometrically (Perkin-Elmer, Lambda 15) according to the method of Omura and Sato (1964). The PROD and EROD activities were determined fluorimetrically in the S9 fraction (Perkin-Elmer, LS-5B), according to the method of Lubet et al.(1990)
.
DNA synthesis (cell proliferation) assays.
Cell proliferation in livers of female mice and in kidneys of male rats was assessed using the 5-bromo-2-deoxyuridine (BrdU) technique, which determines the rate of DNA synthesis. Osmotic minipumps (Alzet osmotic pump, rat: model 2ML; mouse: model 2001), containing a freshly prepared BrdU solution in saline at a concentration of about 20 mg/ml, were implanted subcutaneously in the back region 3 and 7 days prior to necropsy in mouse and rat studies, respectively, under Metofane® (Janssen, Germany) anesthesia. The jejunum served as positive control tissue in cell proliferation determinations in both species.
Necropsy, histotechnique, and immunohistochemistry.
A full necropsy was performed on all animals, except on those used for biochemical examinations. The animals were anesthetized under CO2, weighed, killed by decapitation, exsanguinated, and assessed for the presence of gross lesions. Kidney weights were determined in male rats, and liver weights were determined in female mice. Kidneys and jejunums of male rats as well as livers and jejunums of female mice were fixed in a 4% formaldehyde solution for 2472 h, followed by a 70% ethanol bath, and subsequently embedded in paraplast. Sections of the kidneys (1 longitudinal and 1 transverse), the livers (sections of lobus dexter lateralis and lobus dexter medialis), and the jejunums were prepared. For light microscopy, these sections were stained with hematoxylin and eosin. In addition, Mallory-Heidenhain stain was used to identify hyaline droplets in kidney sections of controls and high-dose rats from the main study. Hematoxylin-eosin slides of liver and kidneys were also used to determine the mitotic index in these tissues.
All sections for immunohistochemistry were dewaxed with xylene and ethanol (100%, 96%, 70%) and subsequently incubated with protease (0.1%) for 1 or 2 min at 37°C.
Liver and kidney slides for the assessment of apoptosis were prepared using Tunel stain (Boehringer) for 60 min at 37°C, followed by labeling with antifluoreszin-conjugated alkaline phosphatase for 30 min at 37°C, then chromogene complexed with fast red for 8 min and counter-stained with hematoxylin (Mayer) for 5 min. A negative control slide was prepared by substituting Tunel reagent for phosphate-buffered saline. The slides were covered with Kaisers glycerol gelatin. A red reaction product covering the nucleus characterized apoptotic cells.
Liver, kidney, and jejunum sections for the assessment of DNA synthesis were hydrolyzed with 4 N HCl for 30 min, incubated with a primary monoclonal mouse anti-BrdU antibody for 24 h at 4°C, then incubated with a biotinylated antimouse link antibody for 20 min, and labeled with an alkaline phosphatase/Streptavidin complex for 20 min, followed by chromogene complexing with fast red for 4 min, and counter-staining with hematoxylin (Mayer) for 5 min. The slides were covered with Kaisers glycerol gelatin. Cells in S-phase were characterized by a red reaction product covering the nucleus.
Kidney sections for the assessment of 2u-globulin accumulation were incubated with a primary mouse anti-
2u-globulin antibody for 24 h at 4 °C, then incubated with a biotinylated antimouse link antibody for 20 min and labeled with an alkaline phosphatase/streptavidin complex for 20 min, followed by chromogene complexing with fast red for 4 min, and counter-staining with hematoxylin (Mayer) for 5 min. The slides were covered with Kaisers glycerol gelatin.
2u was characterized by a red reaction product in the cytoplasm of affected cells.
Quantitative immunohistology.
An image analysis system (Quantimed 500, Leica, or KS 400, Zeiss, Germany) was used to quantitate the proportion of cells engaged in DNA synthesis (BrdU-stained cells).
In the liver, BrdU labeling was evaluated in the whole-liver lobule as well as in the 3 zones of the liver lobule according to Rappaport et al.(1954), which comprise the portal triad region (zone 1), the midzonal region (zone 2), and the central vein region (zone 3). Positively labeled hepatocytes were discriminated from mesenchymal cells on the basis of differences in shape and size. Labeled and unlabeled cells were counted by genuine color detection. With this technique, more than 1000 cells per zone (equal to more than 3000 cells per animal) are evaluated (Bahnemann et al., 1997
; Goldsworthy et al., 1991a
,b
; 1993
). Determination of the mitotic index in hematoxylin-eosin-stained sections was performed likewise. The proportion of apoptotic cells (Tunel-stained cells) was determined by light microscopy.
The evaluation of cell proliferation in the kidneys was performed stepwise according to the method described by Larson et al. (1994). The evaluation of cells in S-phase was restricted to the proximal tubuli of the cortex, since labeling activities were only found here. Proximal tubular cells were distinguished from distal tubular cells on the basis of differences in lumen, cell shape, and cell size. Labeled and unlabeled cells were counted by genuine color detection. The BrdU labeling pattern was not evenly distributed over the whole cortex but was clearly multifocal, forming "hot spots" of cell proliferation in the entire cortex (referred to as "cortex 2," see Fig. 2
). Within the cortex, specifically in the first 4 layers of proximal tubulus underneath the renal capsule (referred to as "cortex 1," see Fig. 3
), the hot spots were seen most often. Each hot spot consisted of at least 5 positively labeled cells in an area of about 80 proximal tubular cells. BrdU labeling was evaluated in 15 hot spots as well in the whole cortex (cortex 2) and in the subcapsular area of the cortex (cortex 1). With this technique, approximately 1200 cells (80 cells each in 15 hot spots) were evaluated in cortex 2 and cortex 1.
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Electronmicroscopy.
Liver tissue (lobus dexter medialis and lateralis) from 5 mice exposed to 0 or 5400 mg/m3 for 20 days, immersion fixed, and stored in 4% formaldehyde solution was used to prepare semithin sections, which were then examined. Midzonal (zone 2) and central vein (zone 3) regions were subsequently selected for the preparation of ultrathin sections, which were then examined for any degenerative changes in cell organelles or subcellular components.
Statistics.
Body weights were analyzed with a parametric one-way analysis using the F-test (ANOVA, two-sided). If the resulting p-value was equal to or less than 0.05, a comparison of each group with the control group, using the Dunnetts test (two-sided), was performed for the hypothesis of equal means (Dunnett, 1955, 1964
; Winer, 1971
). Cytochrome P450, ethoxyresorufin-O-deethylase (EROD), and pentoxyresorufin-O-depentylase (PROD) data were analyzed using the Mann-Whitney test for the hypothesis of equal medians (Siegel, 1956
). A nonparametric one-way analysis, using the Kruskal-Wallis test (two-sided), was applied to organ weight data. If the resulting p-value was equal to or less than 0.05, a pairwise comparison of each dose group with the control group was performed using the Wilcoxon test for the hypothesis of equal medians (Hettmannsperger, 1984
; Miller, 1981
; Nijenhuis and Wilf, 1978
). Immunohistochemistry data were analyzed by pairwise comparison of each dose group, with the control group using the Wilcoxon test (two-sided) for the hypothesis of equal medians (Siegel, 1956
).
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RESULTS |
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Mortality and clinical findings.
There were no deaths in the course of the study. The observed clinical signs did not distinguish treated animals from controls. Body weight development was not affected by treatment (data not shown).
Effects on the kidneys of male rats.
Gross kidney changes were not observed, and there was no effect of THF on kidney weights. THF caused no induction of drug-metabolizing enzymes. Morphological examination of H&E slides of the kidneys revealed no microscopic changes that distinguished treated animals from controls. Apoptosis (or necrosis) was not identified in H&E kidney slides, and mitosis was observed in 3 animals only (one control animal and one high-dose animal after 5 exposures, and one high-dose animal after 20 exposures) (Table 3).
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Evaluation of the hot spots of cortex 1 revealed a slight stimulation of cell proliferation after 5 exposure days at 5400 mg/m3. A significantly higher number of BrdU-labeled cells was recorded after THF exposure at 5400 mg/m3 for 20 days (see Figs. 4 and 5). At 1800 mg/m3, a slightly higher number of BrdU-labeled cells was also recorded in hot spots after 20 days. No effects on cell proliferation were recorded at 600 mg/m3. Cell proliferation was no longer increased in the renal cortex after the three-week recovery period. When evaluating the entire cortex (cortex 2), the results of cell proliferation were less prominent, i.e. an increase was only noted in the 5400-mg/m3 group after 20 exposures. When using the Larson approach, none of the 3 THF concentrations caused statistically significant deviations in the overall BrdU labeling index in the renal cortex at any time.
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Effects on the liver of female mice.
Gross liver changes were not observed at any time. There was no effect on liver weight at any THF dose after 5 exposure days, but slightly higher absolute and relative liver weights were recorded after 20 exposure days at 5400 mg/m3; absolute liver weight controls: 0.96 ± 0.04 g vs. 1.06 ± 0.07g (p < 0.01) in the high dose, relative liver weight controls: 4.19 ± 0.17 vs. 4.43 ± 0.26 (p < 0.05) in the high dose. Significantly higher cytochrome P450 levels and increased EROD and PROD activities were recorded at 5400 mg/m3 after 5 exposure days; P450 (nmol/mg) controls: 0.054 ± 0.09, high dose 0.67 ± 0.06 (p < 0.05); EROD (pmol/min/mg) controls: 30.6 ± 9.1, high dose 58.8 ± 15.0 (p < 0.01) and PROD (pmol/min/mg) controls: 8.0 ± 7.1, high dose 25.7 ± 13.2 (p < 0.05).
Cell proliferation was significantly increased after 5 days of treatment in the high concentration group in zones 2 and 3. An increase was also observed after 20 exposures of the highconcentration in zone 3. This effect was reversible within 3 weeks of recovery. The evaluated numbers of mitotic cells confirmed the data of S-phase response. At 1.800 mg/m3, no relevant effects were recorded in either the BrdU labeling mitotic or apoptotic indexes. At 600 mg/m3, no effects on the liver were recorded. There were no morphological signs of cell degeneration or necrosis, and TUNEL staining revealed no changes in the number of apoptotic cells at 5400 mg/m3 after 5 or 20 exposure days (see Table 4). Electron microscopy did not reveal degenerative changes in cell organelles or subcellular compartments in midzonal or centrilobular hepatocytes after 20 exposure days at 5400 mg/m3.
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DISCUSSION |
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Following the conventional stepwise measurement of cell proliferation in the kidneys according to Larson et al. (1994), no significant changes compared to control animals were obtained. However, during evaluation, it was noted that BrdU-positive cells were not evenly distributed over the renal cortex, but rather accumulated focally. These foci, comprising 5 or more labeled cells, were named hot spots of cell proliferation. They were located in the proximal renal tubules, and were regarded to represent the target cell population. Quantitation of the hot spots was achieved by evaluation of the entire cortex (named cortex 2 in Table 4
) for hot spots. The fist 15 hot spots encountered were recorded in descending numbers of labeled cells. Thereafter, the hot spot with the lowest number of labeled cells was replaced if a hot spot with a higher number of labeled cells was found. This ensured that those hot spots with the highest number of labeled cells were reported and that subjective selection was excluded.
Using this procedure, cell proliferation was significantly increased in cortex 2 at 5400 mg/m3 after 20 exposures.
While measuring cell proliferation with the hot-spot approach in the entire cortex (cortex 2), it became evident that there was a higher number of hot spots in an area directly underneath the renal capsule, just covering the first 4 layers of transverse proximal renal tubules (cortex 1 in Table 4). Using the same approach of measuring cell proliferation in the hot spots in cortex 1 as described for cortex 2, cell proliferation was significantly increased after 5 exposures of 5400 mg/m3 and after 20 exposures at 5400 and 1800 mg/m3.
There were no proliferative effects at the noncarcinogenic dose of 600 mg/m3, even though 2u accumulation was detectable.
There was no evidence of microsomal enzyme induction in male rat kidney, but clear effects were noted in female mouse liver at 5400 mg/m3, accompanied by increased liver weights, and a higher mitotic as well as BrdU labeling index in midzonal and centrilobal regions after 5 and 20 days of exposure. There was no indication of cytotoxicity as the origin of the proliferative responses (there were no degenerative or necrotic effects in liver cells or organelles, and no changes in the number of apoptotic cells). Within the recovery period the effects were largely reversible. Weak proliferative effects were recorded at 1800 mg/m3 after 20 exposures, whereas no effects were noted at 600 mg/m3 (an increased liver tumor incidence in the bioassay was noted at 5400 mg/m3, only). For the (mouse) liver the initial change is most likely an increased cytochrome P450 content, increased ethoxyresorufin-O-deethylase and pentoxyresorufin-O-depentylase activity as a response to increased functional demand. Increased liver cell proliferation and increased mitotic index are a further response to the THF exposure. The fact that there was no increase in the number of apoptotic cells suggests that the changes are related to an adaptation to increased functional demand, rather than THF-related liver toxicity.
The present study demonstrates the induction of cell proliferation by THF in those organs in which an increased incidence of tumors was observed in the chronic studies with rats and mice and provides a mechanism for the increased tumor formation. In the absence of a biologically relevant genotoxic potential it can be argued that at dose levels that do not increase cell proliferation in these target organs there is no carcinogenic risk. The concept of a threshold for THF-mediated carcinogenic effects is an important aspect for risk assessment. Moreover, in the specific cases of male rat kidney tumor formation and B6C3F1 mouse liver tumor formation, there is also some evidence that the nongenotoxic carcinogenic mode of action may not be relevant for humans.
2u-globulin nephropathy is initiated by its accumulation in the phagolysosomes of the proximal convoluted tissue, with subsequent acceleration of apoptosis and replicative cell turn over (Alden, 1991
; Caldwell et al., 1999
). This sequence of changes and the subsequent occurrence of kidney tumors in male rats has been observed with several other chemicals, such as methyl tert-butyl ether (Prescott-Mathews et al, 1997
), 1,4-dichlorobenzene (Lake et al, 1997
), limonene (Turner et al, 2001
), tert-butyl alcohol (Borghoff et al, 2001
), and decalin (Ridder et al, 1990
). A strong association between sustained
2u-globulin accumulation and renal neoplasia has been described by several groups of authors (Baetcke et al., 1991
; Dietrich and Swenberg, 1991
; IARC, 1998
; Short et al. 1989
; Swenberg and Lehmann-McKeeman, 1998
).
2u was shown to cause morphological transformation in the pH 6.7 SHE cell transformation assay; this effect was not achieved by other proteins nor by typical
2u-inducing compounds such as d-limonene or 2,2,4-trimethylpentane (Oshiro et al 1998
). In the evaluation paper prepared for the U.S. EPA risk assessment forum, it is stated that "compounds producing renal tubule tumors in male rats attributable solely to chemically induced
2u-globulin accumulation will not be used for human cancer hazard identification or for dose-response extrapolations," indicating that the
2u-induced kidney tumor formation in male rats is a sex- and species-specific effect (Baetcke et al 1991
).
Other compounds are also known to induce cytotoxicity-related replicative DNA synthesis and cell proliferation as the driving forces for an increase of liver tumors in mice. Enhanced cell proliferation and suppression of apoptosis are typical characteristics of nongenotoxic tumor promoters (Schulte-Herman, 1983). 1,4-Dioxane is a well-investigated example (Goldsworthy et al., 1991a
,b
; Miyagawa et al., 1997
; Stoll et al., 1981; Yamazaki, 1994
), which also has structural similarities with THF. Moreover, non genotoxic chemicals that induce hepatic metabolic enzyme systems and increase liver weights have been frequently observed to increase liver tumor formation in mouse strains, which have a high spontaneous background of liver tumors such as the B6C3F1 mouse (Park et al. 1990). The unusual sensitivity of the B6C3F1 and other sensitive mouse strains has been taken into account in the guidelines for the classification of carcinogenic substances in the European Union, which now separates these substances into 3 different categories: category 1, based on sufficient epidemiological evidence; category 2, in cases of sufficient animal data in two species; and category 3, if the substance is not genotoxic and the nongenotoxic mode of action resulting in tumor formation has been elucidated. An animal carcinogen should not be classified in any of these categories if the mechanism of experimental tumor formation is clearly identified, with good evidence that this process cannot be extrapolated to man, or the only available tumor data are liver tumors in certain sensitive strains of mice. The present study provides some evidence that THF would be a suitable candidate for no classification in the context of the EU classification schemes.
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ACKNOWLEDGMENTS |
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NOTES |
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