Toxicity of Tetrafluoroethylene and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine in Rats and Mice

Douglas A. Keller1, Gerald L. Kennedy, Jr.2, Paul E. Ross3, David P. Kelly and Glenn S. Elliott4

DuPont Haskell Laboratory for Toxicology and Industrial Medicine, P.O. Box 50, 1090 Elkton Road, Newark, Delaware 19714

Received January 18, 2000; accepted April 10, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Groups of 25 female F344 rats and 25 female B6C3F1 mice were exposed to 0, 30, 300, 600, or 1200 ppm tetrafluoroethylene (TFE) by inhalation for up to 12 days. Another set of 25 female rats and 25 female mice of the same strains were given 0, 5, 20, or 50 mg/kg of S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-CYS) by oral gavage for 12 days. Both 12-day exposure regimens consisted of exposures for 5 consecutive days, a weekend with no exposures, and 4 consecutive daily exposures following the weekend. Five animals per group were sacrificed after the first exposure, the fifth exposure, and the ninth exposure for evaluation of cell proliferation in the liver and kidney. The remaining animals in each group (up to 10) were sacrificed after the ninth exposure (test day 12) for pathological evaluation of the liver, kidney, and spleen. Clinical pathology evaluations were performed on test day 11 or 12. Inhalation of TFE by rats and mice caused slight microscopic changes in the kidneys of rats and mice, but no histopathological changes in the liver. In the kidney, administration of TFE-CYS by gavage caused severe microscopic changes in rats, moderate-to-severe changes in mice, and no microscopic changes in the liver. Cell proliferation was increased in the kidneys of rats and mice given TFE by inhalation and TFE-CYS by gavage. TFE-CYS also caused increased liver weights and cell proliferation in the liver of rats and mice at the high doses. The cell proliferation response in the kidney and liver was transient in both species, being most pronounced after 5 days of exposure, and less evident or absent after 12 days of exposure. In the kidney, the cell proliferation and histopathologic response in rats was generally more pronounced than in mice. Kidney damage and cell proliferation were confined to the pars recta (P3) of the outer stripe of the outer medulla and medullary rays. Tubules in mice exposed to TFE and TFE-CYS had mostly regenerating cells by test day 12, while in rats the tubules still showed marked degeneration along with regeneration by the end of the study. The cortical labyrinth (P1 and P2 segments) was also affected at the 50 mg/kg dose of TFE-CYS in rats. Rats exposed to 50 mg/kg TFE-CYS had a mild anemia, and rats exposed to 1200 ppm TFE had slight, biologically inconsequential decreases in erythrocyte mass that may have been compound-related. In spite of the rather pronounced histopathologic changes in the kidneys of rats exposed to TFE-CYS, there was no clinical chemistry evidence for decreased kidney function. Increased levels of urinary fluoride were present in rats exposed to 300 ppm and greater of TFE, and in rats exposed to 20 and 50 mg/kg TFE-CYS. The spleen was not affected in this study. Overall, the results of this study suggest that effects of TFE could be attributed to the toxicity of TFE-CYS over the course of a 2-week exposure, as all effects that were seen with TFE were also seen with TFE-CYS.

Key Words: tetrafluoroethylene; S-(1,1,2,2-tetrafluoroethyl)-L-cysteine; kidney; liver, cell proliferation..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tetrafluoroethylene (TFE) is used as a starting material in a variety of polymer applications. TFE has low acute inhalation toxicity (Kennedy, 1990Go), but upon long-term administration, a variety of tumors were observed in rats and mice (National Toxicology Program [NTP], 1997). A no-observed-effect level for oncogenicity was not established in the long-term studies for mice or for female rats (lowest concentration tested, 312 ppm). In male rats, no statistically significant increase in tumors was present at 156 ppm, but higher concentrations were oncogenic. The significant tumors were renal tubular adenomas and carcinomas in both sexes of rats, hepatic hemangiosarcomas in both sexes of mice and in female rats, and hepatocellular adenomas and carcinomas in both sexes of mice and rats. Histiocytic sarcomas were also observed in many tissues of exposed mice.

It has been well established that TFE causes kidney tubule damage via the processing of a glutathione (GSH) conjugate (Lock, 1988Go; Odum and Green, 1984Go). This GSH-mediated mechanism likely leads to tumor formation by causing a constant regeneration of the epithelium in the kidney and increased cell proliferation, resulting in greater opportunity for mistakes in DNA synthesis, and mutations (Cohen, 1998Go; Cohen and Ellwein, 1990Go). This mechanism would be classified as non-genotoxic and is supported by the current lack of positive mutagenicity data with TFE or TFE-GSH conjugates (Green and Odum, 1985Go). Data are plentiful on the rat, but little or no data are available on GSH conjugation of TFE in mice. While mice exposed to TFE have non-neoplastic kidney damage, no kidney tumors develop, and the metabolic fate of the GSH conjugate is unknown.

No previous studies indicated the potential for TFE to cause liver tumors. The only known route of metabolism for TFE is via GSH conjugation (Odum and Green, 1984Go), but the potential for activation via cytochrome P450 has not been investigated thoroughly. There is no precedent for GSH conjugates causing hemangiosarcomas in the liver, and other vinyl compounds (vinyl chloride, vinyl bromide, vinyl fluoride) that cause hepatic hemangiosarcomas form epoxides via P450-mediated mechanisms (Henschler, 1985Go).

More detailed knowledge of the biological activities of TFE or its metabolites, particularly in the liver, and of any differences between species in the production of the metabolites would aid in extrapolating possible tumor outcomes between species. Mechanistic data would also aid extrapolation to lower concentrations, where rodents were not tested but where worker and community exposures occur, and to short-term, high-exposure situations.

The objective of the study was to determine if the toxicity of TFE in rats and mice could be due to its major metabolite, S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-CYS). Results from this study may indicate whether the cysteine conjugate alone is responsible for the toxicity of TFE or if another metabolite may be involved.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The test materials were tetrafluoroethylene (TFE) and S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFE-CYS). TFE (purity > 99%) was supplied by DuPont. TFE-CYS was synthesized by the method of Odum and Green (1984) by SynQuest, Inc. (Gainesville, Florida), and the structure confirmed by NMR and mass spectrometry (purity > 99%). TFE was a gas at room temperature and was stored as a gas in steel cylinders; d-Limonene was added to each cylinder as a stabilizer. TFE-CYS was an off-white solid and was stored frozen.

Animals
Approximately 225 female F344 rats and 225 female B6C3F1 mice were obtained from Charles River Breeding Laboratories, Raleigh, NC. Animals were ordered separately for the oral and inhalation phases of the study, and arrived at different times. The rats and mice were approximately 49 days of age on arrival. These strains were selected because they were the strains used by the National Toxicology Program (NTP, 1997Go) in the long-term bioassays. Females were used since they were the more sensitive sex in the NTP study.

Inhalation Exposures
Atmosphere generation.
The targeted chamber concentrations were 0, 30, 300, 600, and 1200 ppm. TFE vapor was generated by regulating the TFE tank pressure and metering the test material through stainless steel tubing, and into rotometers. A separate flow meter was used for each test chamber. The vapor was diluted by filtered, conditioned air to the desired concentrations for each of the 4 test chambers. Regulating the flow of the test substance through the flow meters controlled chamber concentrations of TFE. Only filtered, conditioned air passed through the control chamber. Chamber exhausts from the test chambers were discharged through an exhaust stack.

Chamber construction and design.
The exposure chambers were constructed of stainless steel and glass. The internal nominal volume of the chambers was approximately 300 L. Exposure chambers were cubical with square-pyramidal chamber inlets and outlets (NYU style); a tangential feed at the chamber inlet promoted gas mixing and uniform distribution of the test substance vapor. Homogeneous distribution of TFE vapor in the high level chamber was verified during pre-study method development.

Exposure mode.
Each day during the 6-h whole-body exposures, each rat was individually housed in a stainless steel wire mesh cage. Exposures were conducted during approximately the same time period each day to minimize diurnal physiological variations. At the end of each exposure, rats remained in their chambers long enough to allow clearance of the test material from the chamber atmosphere, as determined by atmospheric analysis. Control rats were treated exactly as TFE-exposed rats, except that the chamber atmosphere did not contain the test material. During the exposure phase of the study, all rats were weighed on test days 1, 5, 8, and 12, and individually observed for clinical signs of toxicity before and after each exposure. Group clinical observations were made during exposures. In addition, rats were checked for an alerting response to an auditory stimulus 3 times during each exposure and once after each exposure.

Characterization of Inhalation Chamber Atmosphere
Test substance sampling and analysis.
The atmospheric concentration of TFE was determined by gas chromatography at approximately 30- to 60-min intervals during each 6-h exposure. Gas samples were continuously drawn by a vacuum pump from representative areas of the chambers where rats were exposed.

Chamber atmosphere samples were analyzed for TFE concentration with a Hewlett Packard Model 5880 gas chromatograph equipped with a gas sample loop and flame ionization detector. All samples were chromatographed isothermally at 90°C. Nitrogen was used as the carrier gas and samples were chromatographed on a 20 in. x 1/8 in. (inside diameter) stainless steel column containing 3% OV 101 on Chromosorb W HP, 100/120 mesh. The atmospheric concentration of TFE was determined by comparing the detector response of the chamber samples to that of gas standards with the use of standard curves. The gas standards were prepared prior to each exposure by injecting known volumes of gaseous TFE into gas bags containing known volumes of air.

Environmental monitoring.
Chamber airflow was targeted at approximately 60 L/min, temperature was targeted at 17 to 23°C, relative humidity was targeted at 40 to 60%, and oxygen concentration was targeted to at least 19%. Airflow, temperature, and relative humidity were monitored continually with the Lander Control System's Toxicology Monitoring System and recorded at approximately 15- to 30-min intervals during each exposure. Oxygen was measured with a Biosystems Model 3100R oxygen monitor and recorded 2 times during each exposure.

Oral Gavage Exposures
The body weight of each animal was taken on each day of dosing. TFE-CYS was dissolved in deionized water and administered by intragastric intubation to groups of female F344 rats and female B6C3F1 mice at a dosage of 5, 20, or 50 mg/kg/day. There were 25 animals per group at initiation of dosing. Solutions were prepared fresh for each test group on each day of dosing. Control groups of 25 female rats and 25 female mice were dosed with deionized water only. The rats and mice were dosed 9 times over a 2-week period (weekend excluded) with the following exception: since mortality occurred in some mice in the 0, 20, and 50 mg/kg groups, replacement mice were dosed beginning on test day 2. Individual dosage volumes were calculated using body weights obtained prior to each dosing. The test and control animals were dosed at a volume of 5 ml/kg of body weight. The animals were observed for clinical signs of toxicity on each day of dosing. Observations for mortality and signs of illness, injury, and abnormal behavior were made daily throughout the study.

Cell Proliferation
Three times during the study, on test days –3, 2, and 9, 5 animals per group were implanted with an osmotic minipump (Alza, Palo Alto, CA) for evaluation of cell proliferation. Each animal was anesthetized with isoflurane, and an osmotic minipump loaded with a sterile solution of 5-bromo-3`deoxyuridine (BrdU, Sigma, St. Louis, MO) implanted subcutaneously. Three days after implantation of the minipump (test days 1, 5, and 12) each animal was sacrificed by carbon dioxide asphyxiation and exsanguination. Liver, kidney, and spleen were saved for potential evaluation of cell proliferation. A portion of the duodenum was saved for monitoring of the staining method for BrdU.

Livers and kidneys from each animal sacrificed for cell proliferation were processed to blocks, sectioned, and stained for BrdU using a monoclonal antibody specific for BrdU. One thousand hepatocytes per liver were counted. BrdU labeling of nonparenchymal cells (cells other than hepatocytes) of control and treated livers were subjectively compared. Since the extent of nonparenchymal cell labeling was similar in all groups, counting was not done. As the bulk of the BrdU renal cell labeling in this study was in the outer stripe of the outer medulla and medullary rays (as were the histomorphological changes), 2000 cells were counted in this zone. The duodenum was processed and checked as a positive control for the uptake of BrdU by the animal and the staining procedure.

Clinical Pathology Evaluations
Clinical pathology evaluations were designed to monitor for liver, kidney, and hematopoetic damage. Urine was collected overnight after the last (9th) exposure or dosing from 10 rats per concentration or dosage. Blood was collected immediately prior to sacrifice on test day 12, from 10 rats per concentration or dosage, and on test day 11 and at sacrifice from 10 mice per concentration or dosage. Blood and urine were not collected from the cell-proliferation animals.

After a fast of {approx} 16 h, blood was taken from the orbital sinus of each fasted rat while it was under light carbon dioxide anesthesia. Blood was collected from mice in a similar manner, but mice were not fasted. Blood collected from mice at sacrifice was taken from the vena cava, not from the orbital sinus. The following hematologic parameters were measured or calculated:

Absolute values for the various types of leukocytes were calculated from the leukocytic data. Blood cell counts, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were determined on a Serono Baker 9000® hematology analyzer. Differential cell counts were determined on a Hematrak® Automated Differential System cell counter. Reticulocyte smears were prepared but evaluation was not needed to clarify hematological findings.

In conjunction with hematological measurements, serum chemical parameters were measured or calculated from each rat. Selected parameters (*) were measured for mice.

Clinical chemical parameters were measured on a Boehringer Mannheim/Hitachi 717 clinical chemistry analyzer using Boehringer Mannheim reagents. Serum globulin concentration was calculated from the total protein and albumin concentrations. Parameters measured in rat urine were volume, glucose, osmolality, protein, specific gravity, and fluoride. Osmolality was determined on a Precision Systems model Multi-OsmetteTM 2430 osmometer. Specific gravity was determined by refractometry. Urine glucose and protein were measured on a ClinitekTM 200 urine chemistry analyzer using Ames MultistixTM urine chemistry dipsticks. Urine fluoride concentration was measured using an ion-specific electrode (Beckman {phi}TM 12 pH/ISE meter).

Pathological Evaluations
Female rats and mice were sacrificed by carbon dioxide anesthesia and exsanguination and necropsied. The liver, kidneys, and spleen of sacrificed rats and mice were weighed at necropsy. Each rat and mouse was given a complete gross examination and representative samples of liver, kidneys, spleen, and sternum were fixed in 10% neutral buffered formalin. Processed tissues from all animals were embedded in paraffin, cut at a nominal thickness of 5 µm, placed on glass slides, and stained with hematoxylin and eosin (H&E). Sections of liver, kidneys, and spleen from the control and treated rats and mice were examined by light microscopy.

Statistical Analyses
Body weights, body weight gains, organ weights, and clinical laboratory evaluations were analyzed by a one-way analysis of variance. When the corresponding F-test for differences among test groups was significant, pairwise comparisons between test and control groups were made with Dunnett's test. Bartlett's test was used to test for homogeneity of variances and significance was judged at p < 0.005.

For clinical pathology data, a 1-way analysis of variance (ANOVA) and Bartlett's test were calculated for each sampling time. Dunnett's test was used to compare means from the control groups and each of the groups exposed to TFE or TFE-CYS. When the results of the Bartlett's test were significant (p < 0.005), the Kruskal-Wallis test was employed and the Mann-Whitney U test was used to compare means from the control groups with each of the groups exposed to TFE or TFE-CYS.

Significance of tests other than Bartlett's were judged at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhalation chamber data.
The daily exposure chamber mean TFE concentrations for 6 hours of exposure were close to or exactly as targeted. The mean chamber concentrations for the exposure period were 0, 31 ± 1, 300 ± 4, 600 ± 3, and 1200 ± 7 ppm. There was a brief excursion to 340 ppm in the 30-ppm chamber on the first day of the study, due to poor control of the very low TFE flow needed for this chamber. The daily mean chamber temperatures for the 4 chambers during this study was 24°C, the daily mean chamber relative humidity ranged from 43 to 45%, and the daily mean airflow ranged from 56 to 64 l/min.

Body weights and clinical observations.
There were no significant effects on body weights or clinical signs in rats or mice exposed to TFE. Female rats dosed at 50 mg/kg TFE-CYS lost weight on most days of dosing. Overall, the 50 mg/kg rats had a mean body weight loss of 17.6 grams for the 12-day dosing period. Rats in the 20 mg/kg group had slight, but not statistically significant body weight decreases over the course of the study. Mice dosed with TFE-CYS did not have statistically significant decreases in mean body weight gains, although there was a trend towards lower body weights in all groups of mice dosed with TFE-CYS (data not shown). Some mice in each TFE-CYS dosage group were accidentally killed, due to dosing trauma. These deaths occurred mainly in the first 6 days of the study, and ranged from 2 to 6 mice per group.

The only clinical observation in rats that could be attributed to the dosing with TFE-CYS was ruffled fur. There were 2 clinical observations in mice that could be attributed to TFE-CYS; mice in the 50 mg/kg group had a higher incidence of hunched posture and lethargy.

Clinical Pathology
Hematology.
Rats exposed to 1200 ppm of TFE by inhalation had minimal decreases in the indicators of circulating erythrocyte mass (RBC, Hb, Ht; Table 1Go). However, the magnitude of these changes was minimal and therefore, biologically inconsequential. The mean values and most individual animal values for RBC, Hb, and Ht were within or above the range of values expected in female rats of this age. Furthermore, the mean values were only 6 or 7% below the control group mean values. Nevertheless, the erythrocyte effects may have been test substance-related, because similar changes of larger magnitude occurred in rats exposed to TFE-CYS by gavage.


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TABLE 1 Hematological Effects and Urinary Fluoride Excretion of TFE and TFE-CYS Exposed Rats
 
Rats exposed to 50 mg/kg of TFE-CYS by gavage had mild anemia. The indicators of circulating erythrocyte mass (RBC, Hb, Ht) were decreased by 11% to 13% compared to controls (Table 1Go). Erythrocyte indices (MCV, MCH, MCHC) were unchanged, indicating that the mild anemia was normocytic normochromic. However, there were no histopathologic changes that are commonly seen in the presence of anemia, such as increased hemosiderin pigment or increased extramedullary hematopoiesis.

In previous studies with TFE, a similar mild normocytic normochromic anemia occurred in female F344/N rats exposed to 5000 ppm by inhalation for 13 weeks (NTP, 1997Go). However, in a 16-day inhalation study with TFE, female F344/N rats did not have anemia at concentrations up to 5000 ppm (NTP, 1997Go).

Clinical chemistry.
No test substance-related, toxicologically important clinical chemistry changes occurred in rats or mice exposed to TFE by inhalation or to TFE-CYS by gavage.

Urinalysis.
No statistically significant changes in urine analytical parameters occurred in female rats exposed to TFE. Mean urine fluoride excretion was increased in females exposed to TFE at 300 ppm and above (Table 1Go). These changes in urine fluoride excretion reflect absorption and metabolism of TFE with release of fluoride and excretion in the urine. The urine fluoride changes were not considered to be biologically adverse.

No biologically adverse changes in urine analytical parameters occurred in female rats exposed to TFE-CYS. There was a slight (< 2-fold) increase in urine volume in rats dosed with 20 and 50 mg/kg TFE-CYS (data not shown). Mean urine fluoride excretion was increased in females exposed to TFE-CYS at 20 mg/kg and above (Table 1Go). These changes in urine fluoride excretion reflect the slightly higher urine output, as well as absorption and metabolism of TFE-CYS with release of fluoride and excretion in the urine. The urine fluoride changes were not considered to be biologically adverse.

Cell Proliferation
There were no biologically significant effects on cell proliferation in rats or mice in the TFE or TFE-CYS groups after a single exposure (test day 1). Inhalation of TFE caused a statistically and biologically significant increase (9-fold over control) in the mean labeling index of the kidney in the 1200-ppm rats on test day 5 only (Fig. 1aGo). The labeling index in the 1200-ppm group was slightly higher than controls on test day 12, but the increase was probably not biologically significant. There were no significant changes in hepatocyte cell proliferation in rats exposed to TFE for 1, 5, or 12 days; all labeling indices were <= 1 (data not shown).



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FIG. 1. Cell proliferation in the kidneys of rats (A) and mice (B) exposed to TFE. Rats and mice were exposed to TFE by inhalation for 1, 5, or 12 days, then evaluated for cell proliferation. The labeling index is the number of labeled cells per 100 cells counted, and is the mean ± SD of 5 animals per group. *Indicates a statistically significant difference from control group at the same time point, by Dunnett's test (p < 0.05).

 
Mice exposed to 600 and 1200 ppm TFE had significantly increased labeling indices in the kidney on test day 5 (Fig. 1bGo). There was no longer a significant effect on cell proliferation in the kidney by test day 12. Hepatic cell proliferation was not affected by TFE exposure (data not shown).

Rats dosed with 20 and 50 mg/kg TFE-CYS had significantly increased cell proliferation rates in the kidney on test days 5 and 12 (Fig. 2aGo). On test day 5, the increases in labeling index were 9-fold and 5-fold over control for the 50- and 20 mg/kg groups, respectively. The labeling index in the kidney of the 5 mg/kg group was slightly increased, but the increase was not statistically significant and was less than 2-fold over control. On test day 12, the labeling indices in the kidney were still significantly increased over control for the 20 and 50 mg/kg groups, but were less than the day-5 values. In the liver, rats dosed with 50 and 20 mg/kg had significantly increased hepatocyte-labeling indices on test day 5 (Fig. 2cGo). The increases over control were 3-fold at 50 mg/kg and 2-fold at 20 mg/kg. There was no apparent increase in labeling of endothelial cells. By test day 12, the labeling indices in the liver had returned to control values.



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FIG. 2. Cell proliferation in the kidneys of rats (A) and mice (B) and in the livers of rats (C) and mice (D) exposed to TFE-CYS orally for 1, 5, or 12 days, then evaluated for cell proliferation. The labeling index is the number of labeled cells per 100 cells counted, and is the mean ± SD of 5 animals per group. *Indicates a statistically significant difference from control group at the same time point, by Dunnett's test (p < 0.05).

 
Mice dosed with TFE-CYS had increased cell proliferation in the kidney at all doses tested on test day 5 (Fig. 2bGo). The increases were 11-fold, 11-fold, and 7-fold over control at the 50, 20, and 5 mg/kg doses, respectively. By test day 12, the labeling indices in the kidney were still significantly increased over control, but were less than the day-5 values. In the liver, mice dosed with 50 mg/kg TFE-CYS had a 10-fold increase in hepatocyte labeling on test day 5 (Fig. 2dGo). By test day 12, the labeling index at 50, 20, and 5 mg/kg were significantly decreased.

The cell proliferation in the kidney was primarily in the outer stripe of the outer medulla and the medullary rays. These locations of cell proliferation were consistent with the lesions seen in the histopathology portion of the study. In the liver, the labeled cells were randomly distributed, with no zonal pattern apparent. No increased labeling of hepatic endothelial cells was noted at any time point.

Organ Weights
TFE inhalation.
Mean absolute and relative (to body weight) kidney weights of rats at the 600- and 1200-ppm levels and the mean absolute and relative liver weights of the 600-ppm rats were statistically significantly higher than those of control rats (Table 2Go). Minimal microscopic lesions were present in kidneys at these 2 levels as well.


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TABLE 2 Mean Absolute Organ Weights
 
The mean relative liver weight of the 300-ppm mice was statistically heavier than controls, but this result was not considered biologically meaningful because of lack of dose response and the absence of corroborative histologic findings.

TFE-CYS gavage.
The mean absolute and relative kidney weights of the 50 , 20, and 5 mg/kg rats were significantly higher than controls (Table 2Go). Microscopic effects were also found in kidneys at these dose levels. In addition, the mean absolute and relative liver weights of the 50 and 20 mg/kg rats, and the 50 mg/kg mice were significantly greater than those of controls. Although there were no correlative microscopic findings associated with these heavier livers, the positive hepatic cell proliferation findings may suggest that the liver weight was affected by treatment. The mean absolute and relative spleen weights of the 50 mg/kg rats were significantly lower than controls. However, microscopic lesions were not present in these spleens. Lower spleen weight was likely a result of lower body weight.

Microscopic findings.
There were no treatment-related microscopic changes in liver or spleen of mice or rats.

The degenerative and regenerative response of the kidneys of rats and mice by both modes of exposure was consistent. Lesions were anatomically restricted to the outer stripe of the outer medulla (OSOM) and medullary rays (except for severe lesions in which cortical labyrinth involvement was seen). The lesions were graded as follows:

Rats (TFE inhalation).
Minimal treatment-related microscopic effects were present in kidneys at the 1200- and 600-ppm levels. In kidneys of 1200-ppm rats, degeneration/necrosis of occasional tubule epithelial cells was manifest as cells that were enlarged, rounded, and had marked vacuolation of cytoplasm. Nuclei were pyknotic (Fig. 3Go). Kidneys of 600-ppm rats had similar changes, though to a lesser extent.



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FIG. 3. Microscopic section from a kidney of a female rat exposed to 1200 ppm TFE by inhalation for 9 exposures. Vacuolar degeneration and nuclear pyknosis characterizes occasional tubular epithelial cells (arrowheads) at the tip of a medullary ray.

 
Mice (TFE inhalation).
Minimal treatment-related microscopic effects were present in kidneys at the 1200-ppm level. In kidneys of 1200-ppm mice, individual cell necrosis was manifest as occasional tubule epithelial cells that were shrunken and rounded, with deeply eosinophilic cytoplasm. Nuclei were pyknotic. Very slight karyomegaly and cytoplasmic basophilia were noticeable in the same region.

Rats (TFE-CYS gavage).
Treatment-related microscopic effects were seen in kidneys of the 5, 20, and 50 mg/kg rats. All ten 50 mg/kg rats had severe vacuolar degeneration and regeneration (Fig. 4Go). For the most part, tubules in the OSOM tended to be more regenerative and tubules towards the tips of medullary rays tended to be more degenerative. But, it was common for any given tubule to have both degenerative and regenerative cells. Degeneration/necrosis of tubule epithelial cells was manifest as cells that were enlarged, rounded, and had marked vacuolation of cytoplasm. Nuclei were pyknotic. Remnants of cytoplasmic and nuclear debris were present in tubule lumens. Cells that were often larger or smaller than mature tubule cells lined regenerating tubules, and their cytoplasm was basophilic. Density of nuclei per unit length was increased and mitotic figures were evident.



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FIG. 4. Microscopic section from a kidney of a female rat exposed to 50 mg/kg TFE-CYS by gavage for 9 exposures. Vacuolar degeneration and nuclear pyknosis characterizes many tubular epithelial cells (arrowheads) in the medullary ray and in the adjacent cortical labyrinth (arrows). The basophilic tint of tubules in the medullary rays indicates regeneration.

 
At 20 mg/kg, degenerative and regenerative lesions, as described above, were moderate in 9 rats and severe in 1.

At 5 mg/kg, minimal degeneration/necrosis of occasional tubule epithelial cells was manifest as described above. Occasional regenerating tubules were lined by cells with slight cytoplasmic basophilia and a few mitotic figures.

Mice (TFE-CYS gavage).
Treatment-related microscopic effects were seen in kidneys of the 5, 20, and 50 mg/kg mice. In 50 mg/kg mice, of the mice that died early, 1 had severe and 5 had moderate renal lesions. In the surviving mice, 4 had moderate and 1 had mild renal lesions. Three mice had moderate-to-severe coagulation necrosis of tubules. Tubules in these areas were dilated and lined by a deeply acidophilic layer, devoid of nuclei. In the papilla, tubule-lining cells were normal, but lumens contained much eosinophilic fluid and occasional aggregates of mineralized debris, reflecting the upstream injury. Regeneration was moderate in 7 mice and mild in 1. Cells larger or smaller than mature tubule cells often lined regenerating tubules, and their cytoplasm was basophilic. Density of nuclei was increased and mitotic figures were evident.

At 20 mg/kg, 1 mouse (an early death) had mild coagulation necrosis of tubules and 11 had moderate regeneration of tubules as described above.

At 5 mg/kg, 2 mice (early deaths) had minimal coagulation necrosis of tubules, 8 had minimal regeneration of tubules, and 2 had mild regeneration of the tubules as described above.

Study Overall
In rats, renal lesions were characterized by vacuolar degeneration/necrosis and regeneration at the 50, 20, and 5 mg/kg levels. In mice, renal lesions were predominately regenerative at the 50, 20, and 5 mg/kg levels. The lesion grade of rats was one higher than that of mice at each exposure level, because of ongoing degeneration in the rat kidneys.

The NOEL for pathology, based on renal histopathology, for female mice exposed to TFE by inhalation, was 600 ppm and for female rats exposed to TFE by inhalation, was 300 ppm. A NOEL for female mice and rats exposed to TFE-CYS by gavage was not determined, as microscopic renal lesions were detected at all levels tested.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inhalation of TFE by rats and mice caused slight microscopic changes in the kidneys of rats and mice, but no histopathological changes in the livers. Administration of TFE-CYS by gavage caused severe kidney lesions in rats, moderate-to-severe kidney lesions in mice, and no histopathological changes in the livers. Cell proliferation was increased in the kidneys of rats and mice given TFE by inhalation and TFE-CYS by gavage. TFE-CYS also caused an increase in cell proliferation in the livers of rats and mice at the high doses. The cell proliferation response in the kidney and liver was transient in both species, being most prominent after 5 days of exposure, and less prominent or absent after 12 days of exposure. In the kidney, the response in rats, as indicated by cell proliferation and histopathology, was generally more severe than in mice. Degenerative and regenerative renal tubular lesions, produced in rats and mice by inhalation exposure to TFE and gavage exposure to TFE-CYS, were consistently in the OSOM and medullary rays. This zonal effect is suggestive of injury to the pars recta (S3, P3) of the proximal tubule (Owen, 1986Go). At the 50 mg/kg level in rats, the lesion also affected the P2 and, most likely, P1 segments of proximal tubules of the cortical labyrinth. In the TFE-CYS portion of the study, the lesion grade of rats was one higher than that of mice at each exposure level, because of ongoing degeneration in the rat kidneys. In the TFE portion of the study, 600-ppm rats had microscopic lesions, whereas 600-ppm mice did not.

The initial microscopic change noted in renal tubule cells of rats was marked by vacuolar change and enlargement and rounding of the cell, compatible with hydropic degeneration (Owen, 1986Go). Up to a point, this is a reversible perturbation of cell membranes that allows more water than usual to enter the cell. It is possible, however, to have too much influx of water, at which point the cell cannot survive. Such appears to be the case in this study. Degeneration of renal tubule cells in mice was not as striking as in the rats. In mice, only occasional shrunken acidophilic cell remnants lined tubules or were present in tubule lumens. A regenerative appearance of tubules was more the rule. Under the conditions of this study, the mouse kidney did not seem to be as sensitive as the rat kidney to exposure to TFE by inhalation or TFE-CYS by gavage. In spite of the rather pronounced histopathologic changes in the kidneys of rats exposed to TFE-CYS, there was no clinical chemistry evidence for decreased kidney function.

The increased liver weights in mice exposed to 50 mg/kg TFE-CYS and rats exposed to 20 and 50 mg/kg TFE-CYS may be due to a subtle increase in size of hepatocytes and/or an increased cell number as a result of the increased cell proliferation in these livers. The spleen was not a target organ in this study.

In rats, kidney effects were not seen after the first exposure to TFE, but by the fifth exposure, the damage was severe enough to cause a 9-fold increase in cell proliferation at 1200 ppm, and, in mice, a 5-fold increase in cell proliferation at 1200 ppm. This cell proliferation effect was almost completely attenuated by the twelfth day of exposure, as shown by the lack of a statistically significant increase in the rat and mouse kidney, and also by the presence of fairly well regenerated tubules in rats and mice. It is apparent that rats and mice can adapt well to the continued exposure to TFE with respect to overt damage to the kidney. Nevertheless, the early damage may eventually lead to the formation of tumors. The metabolism of TFE to TFE-CYS and mercapturic acids has been well-described (Commandeur et al., 1989Go; Odum and Green, 1984Go). Ultimately, it is felt that the TFE conjugate is processed to a thionacyl fluoride (Commandeur et al., 1989Go) that can acylate proteins in the mitochondria of cells (Hayden and Stevens, 1990Go). The lack of mutagenicity observed with the TFE-CYS conjugate (Green and Odum, 1985Go) suggests that the cytotoxicity of the thionacyl fluoride causes massive regeneration of kidney tubule cells, which could lead to a random set of mutations and eventually the formation of adenomas. This type of mechanism for renal tumor formation would be highly dependent on the rate of cell proliferation in the kidney (Cohen, 1998Go; Cohen and Ellwein, 1990Go), and would be dose dependent.

The amount of TFE-CYS formed during TFE inhalation in rats and mice is not known. Hence, the blood levels of TFE and its metabolite that were achieved during the present study are unknown. It is almost certain, however, that the amounts of TFE-CYS given to rats and mice in this study were higher than the amounts of the metabolite formed in animals exposed to TFE. Green et al. (1998) performed a comparative study of TFE and TFE-CYS metabolism in rats and mice. The identity of TFE and TFE-CYS metabolites were identical in the two species, although a quantitative analysis of the metabolism was not performed. Rats and mice both metabolized TFE to difluoroacetic acid, S-(1,1,2,2-tetrafluoroethyl)-N-acetylcysteine, and N-fluoroacetylated metabolites of the cysteine conjugate. Dosing of rats and mice with TFE-CYS confirmed that all detectable TFE metabolites were formed via the cysteine conjugate. Although the qualitative metabolism of TFE and TFE-CYS is identical in rats and mice, the quantitative aspects of the metabolism require further study and may be an important factor in the susceptibility of rats and mice to different tumor types when exposed to TFE.

TFE-CYS caused more severe kidney effects than did TFE. This is expected, based on the previous work with TFE-CYS in single-exposure experiments and the known ability of TFE-CYS to cause kidney tubule damage (Commandeur et al., 1988Go; Odum and Green, 1984Go). No kidney-cell proliferation was apparent in rats or mice after the first exposure to TFE-CYS. By the fifth exposure, there was a marked response in the kidneys of both species, with increased cell proliferation at all doses, and increased cell proliferation was still present on test day 12. Also, the mice accidentally killed before the end of the study had more severe kidney lesions than the mice sacrificed on test day 12. These results show the beginning of an adaptive response in the kidney to TFE-CYS, although the damage is still severe. The more severe response of the rat kidney compared to the mouse kidney may be due to the ability of the mouse to repair damage or adapt to damage faster than the rat, as would be shown by the faster attenuation of the response in mice.

There was a statistically significant increase in the cell proliferation in the liver of rats and mice given 50 mg/kg of TFE-CYS, and in rats given 20 mg/kg TFE-CYS. These increases in cell proliferation were confined to the hepatocytes, and were not apparent in endothelial cells. The effects were transient, being present only on test day 5. On test day 12 the hepatic cell proliferation in mice was significantly decreased compared to controls. The increased cell proliferation in the first 5 days of the study may be associated with the liver weight increases seen on test day 12, since there was not an obvious increase in cell death in the animals examined.

The clinical chemistry and hematology data demonstrate that TFE and TFE-CYS did not have severe effects on the liver and the hematopoietic system in this 2-week study. The effects on erythrocytes in the 1200-ppm TFE rats and the 50 mg/kg TFE-CYS rats are consistent with the data from the NTP 13-week study reported previously. The increased urinary fluoride excretion in the rats exposed to 300 ppm and greater of TFE indicates that metabolism of TFE to liberate fluoride ion occurs. The data also show no difference between the amounts of urinary fluoride excreted at concentrations of 300 ppm and greater, suggesting that metabolism may be saturated at 300 ppm in rats. The increased urinary fluoride excretion in rats given TFE-CYS at 20 and 50 mg/kg indicates processing of the conjugate via a reaction that liberates fluoride (Commandeur et al., 1989Go), and suggests that this process is not saturated at 50 mg/kg. It is unlikely that the kidney toxicity is due to inorganic fluoride, as the lesions seen in the present study are consistent with those observed with single doses of TFE-CYS (Lock and Ishmael, 1998Go), and the metabolism of TFE-CYS is not extensive (Commandeur et al., 1989Go).

It is interesting to note that there were no significant changes in clinical chemistry, despite the severe kidney damage in rats and mice exposed to TFE-CYS. This may be partly due to the fact that the major kidney damage occurred in the first few days of exposure (as seen in the mice that died early and the cell-proliferation animals), while the clinical pathology samples were taken after the last dose. By this time much of the damage had been or was in the process of being repaired. Also of note is that many of the markers used, such as BUN, creatinine, and phosphate are indicators of kidney function rather than damage. Apparently, damage must be extremely severe to cause a measurable disturbance in kidney function.

ß-lyase activity has been shown to be present in the liver as well as in the kidney (Commandeur et al., 1991Go). Therefore, the formation of the thionacyl fluoride intermediate postulated to be responsible for cytotoxicity in the kidney (Commandeur et al., 1989Go) would likely be formed in the liver as well. This reactive metabolite maybe related to the cell proliferation effect in the liver, although the lack of mutagenicity by TFE-CYS (Green and Odum, 1985Go) suggests that this metabolite is not directly linked to the hemangiosarcoma formation.

Lock and Ishmael (1998) and Commandeur et al. (1988) investigated the hepatotoxicity of a single dose of TFE-CYS, at similar doses to those used in the present study, and found no evidence for hepatic effects. It is evident from those studies as well as the present study, that the hepatic effects of TFE-CYS are subtle enough to require multiple doses for the effect to become evident. More sophisticated techniques will be required to determine the role, if any, of metabolites in the hepatocarcinogenesis of TFE. At the present time, there is no evidence for a genotoxic mechanism in the development of renal or hepatic tumors from exposure of animals to TFE. Further research will be required to fully investigate the mechanisms of oncogenicity related to TFE exposure, and the relevance of the NTP results to humans.

Overall, the results of this study suggest that effects of TFE could be attributed to the toxicity of TFE-CYS over the course of a 2-week exposure. All effects that were seen with TFE were also seen with TFE-CYS. This is the first repeated-dose study reported with TFE-CYS, and the cell proliferation effects in the liver suggest that TFE-CYS may have toxic activity outside of the kidney.


    ACKNOWLEDGMENTS
 
This work was funded by the Association of Plastics Manufacturers in Europe (APME). The authors thank D. Farrar, R. Jung, and G. Malinverno for their useful comments.


    NOTES
 
1 Present address: Sanofi-Synthelabo Pharmaceuticals, 9 Great Valley Parkway, Malvern, PA 19355. Back

2 To whom correspondence should be addressed. Fax: (302) 366–5207. E-mail: gerald.l.kennedy{at}usa.dupont.com. Back

3 Present address: MPI Research, Mattawan, MI 49071. Back

4 Present address: Sierra Biomedical, Inc., 587 Dunn Circle, Sparks, NV 89431. Back


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 TOP
 ABSTRACT
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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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