Rapid Uptake, Metabolism, and Elimination of Inhaled Sulfuryl Fluoride Fumigant by Rats
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《毒物学科学杂志》
Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan 48674
Dow AgroSciences LLC, Indianapolis, Indiana 46268
ABSTRACT
Sulfuryl fluoride (SO2F2) is a structural fumigant gas used to control drywood termites and wood-boring beetles. The pharmacokinetics and metabolism of inhaled SO2F2 were evaluated in male Fischer-344 rats exposed to 30 or 300 ppm 35S-labeled SO2F2 for 4 h. Blood, urine and feces were collected during and after the exposures and analyzed for radioactivity, 35S-labeled fluorosulfate and sulfate, and fluoride (urine and feces only). Selected tissues were collected 7 days post-exposure and analyzed for radioactivity. During and after unlabeled SO2F2 exposures, blood, brain, and kidney were collected and analyzed for fluoride ion. SO2F2 was rapidly absorbed, achieving maximum concentrations of radioactivity in both plasma and red blood cells (RBC) near the end of the 4-h exposure period. Radioactivity was rapidly excreted, mostly via the urine. Seven days post-exposure, small amounts of radioactivity were distributed among several tissues, with the highest concentration detected in respiratory tissues. Radioactivity associated with the RBC remained elevated 7 days post-exposure, and highly perfused tissues had higher levels of radioactivity than other non-respiratory tissues. Radioactivity cleared from plasma and RBC with initial half-lives of 2.5 h after 30 ppm and 1–2.5 h after 300 ppm exposures. The terminal half-life of radioactivity was 2.5-fold longer in RBC than plasma. Based on the radiochemical profiles, there was no evidence of parent 35SO2F2 in blood. Identification of fluorosulfate and sulfate in blood and urine suggests that SO2F2 is hydrolyzed to fluorosulfate, with release of fluoride, followed by further hydrolysis to sulfate and release of the remaining fluoride.
Key Words: sulfuryl fluoride; fluorosulfate; fluoride; inhalation; rat.
INTRODUCTION
Sulfuryl fluoride (SO2F2, VIKANE gas fumigant) is a colorless, odorless gas that has been used to fumigate buildings since 1961 to control pests, including drywood termites and wood-boring beetles. The American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value and OSHA permissible exposure level for SO2F2 are 5 ppm TWA and 10 ppm STEL (ACGIH, 1998). Extensive toxicological studies have been conducted with SO2F2 over the past 30 years, and the toxicity is well characterized (reviewed in Nitschke and Eisenbrandt, 2001). SO2F2 is acutely toxic to mammals when inhaled, with a LC50 (4 h) for rats of about 1000 ppm. Rats exposed to SO2F2 at concentrations of 1500 ppm or 2000 ppm showed central nervous system depression (sedation) within 20 to 40 min, and continued exposure resulted in convulsions and adverse effects on the respiratory tract, liver, and kidneys, followed by death within 3–4 h (unpublished data of The Dow Chemical Company). Exposure of rats to 4000, 10,000, 20,000, or 40,000 ppm SO2F2 decreased the time to incapacitation and death as the vapor concentration increased (Nitschke et al., 1986).
Fluoride ion may play a role in the mechanism of action of SO2F2 in insects and mammals. Many of the observations in rodents overexposed to SO2F2 are typical of acute fluoride poisoning (Drill, 1954; Goodman et al., 1980; Greenwood, 1940; Whitford, 1996). Eisenbrandt and Nitschke (1989) studied the toxicity of SO2F2 in rats and rabbits exposed to 0, 30, 100, or 300 ppm for 6 h/day, 5 days/week for 13 weeks. A statistically significant and dose-related increase in ionic fluoride concentration was reported in the serum of rabbits exposed to SO2F2 for 13 weeks, although serum levels of ionic fluoride were not significantly elevated in rats. Inhalation exposure of rats to 300 ppm SO2F2 resulted in decreased body weights; histopathological changes in the respiratory tract, brain, and kidneys; as well as mottled teeth and decreased specific gravity of the urine. At 100 ppm, the tooth enamel changes were the only exposure-related changes in the rats. No treatment-related effects were observed at 30 ppm. Neuropathological changes in the rats consisted of minimal vacuolation in the area of the caudate-putamen nuclei that was more prominent in the white fiber tracts of the internal capsule than in the adjacent neuropil. Alteration of neurological function in the rats exposed to 300 ppm SO2F2 for 13 weeks was demonstrated by a decreased flicker fusion threshold, and a slowing of flash, auditory, and somatosensory evoked potentials (Mattsson et al., 1988). The functional responses as well as brain histology were within normal limits at 2 months post-exposure.
Mottled incisor teeth in rats exposed to 100 or 300 ppm SO2F2 for 13 weeks were consistent with an increase in systemic fluoride as produced by several other inorganic and organic fluoride compounds (Eagers, 1969; Pattison, 1959). The current metabolism and disposition study was undertaken to better understand the mammalian toxicity of SO2F2 and to determine the systemic availability of fluoride following SO2F2 exposure.
MATERIALS AND METHODS
Test material.
Sulfuryl fluoride, radiolabeled with 35sulfur, was obtained from Moravek Biochemicals, Inc., Brea, California with a specific activity of 10 mCi/mmol. A radiochemical purity of 100% 35SO2F2 was established using gas chromatography with flow radiogas monitoring and identification by retention time confirmation using an authentic standard of SO2F2 and thermal conductivity detection. Structural confirmation was accomplished by gas chromatography/electron impact ionization/mass spectrometry. Non-radiolabeled SO2F2 was obtained from Dow AgroSciences LLC (Indianapolis, IN). Analysis of this test material prior to the study revealed the sample to be 99.8% SO2F2. All other chemicals and solvents were reagent grade or better.
Animals.
Male Fischer-344 rats (197–242 g) were used because they were the same strain used in previous inhalation toxicity studies of SO2F2 (Eisenbrandt and Nitschke, 1989; Mattsson et al., 1988; Nitschke et al., 1986). Upon arrival at the laboratory, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International), each animal was evaluated by a laboratory veterinarian and found to be in good health and acceptable for study purposes. The rooms in which the animals were housed had the relative humidity maintained within a range of 40–70% and the room temperature maintained at 22° ± 3°C. An approximate 12 h light/dark photocycle was maintained for all animal rooms. Room air was exchanged 12–15 times/h. On each of 2 days prior to exposure, the animals were stage-acclimated to the nose-only chamber (1 h on day 1, 2 h on day 2), with the nose cones connected to the exposure chamber and airflow comparable to what it was during exposure.
Rats cannulated in the jugular vein by the supplier (Hilltop Lab Animals, Inc. Scottsdale, PA) were used for the 35S time-course experiments. To increase the likelihood that the cannulae remained patent, acclimation of the rats to the laboratory environment was limited to 4 days prior to 35SO2F2 exposure. They were returned to their cages for 24 h before 35SO2F2 exposure, and during this period the rats underwent exteriorization of the cannulae under light isoflurane anesthesia, approximately 18 h prior to 35SO2F2 exposure.
Non-cannulated rats used for the remainder of the study were obtained from Charles River Laboratories (Raleigh, NC). The animals used for 35S disposition were acclimated to metabolism cages for at least 2 days. They were returned to the metabolism cages for 24 h before 35SO2F2 exposure. Non-cannulated animals used for fluoride analysis were housed one per cage in stainless-steel cages. Non-cannulated animals were selected from those available and assigned to exposure groups using a computer-driven randomization procedure.
Following exposure to 35SO2F2, non-cannulated animals were housed one per cage in glass metabolism cages. Air was drawn through the metabolism cages at 500 ml/min. Cannulated animals were housed in plastic tubs with corncob bedding for the duration of blood collection. Animals exposed to non-radiolabeled SO2F2 were housed one per cage in stainless-steel cages containing a feed container and a pressure activated nipple-type watering system. Cages were suspended above catch pans, and feed and water were provided ad libitum except during the exposures. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO) in pelleted form. To minimize the animals' exposure to environmental fluoride, the animals were given Milli-Q de-ionized water (Millipore Co., Bedford, MA) after their arrival at the laboratory. No attempt was made to remove/reduce fluoride ion from nonaqueous sources, i.e., feed (13 ppm fluoride ion, Nutritional Analyses of Certified Rodent Diet #5002 by PMI Nutrition International).
Exposure system.
The 35SO2F2 exposures were conducted in a 2-liter Plexiglas nose-only exposure chamber, stationed in a closed, vented, Plexiglas box, which was located inside a secondary vented area. The rats were held in place by use of a tubular restraint device constructed from #3 mesh galvanized hardware cloth (7.26 mm openings) attached to a polycarbonate nose cone. An excreta separator-collector was attached to the bottom of the restraint device, and the collectors were placed in dry-ice. Chambers used for the non-radiolabeled exposures consisted of a 42-liter Dow-modified ADG nose-only chamber (ADG Developments, Ltd., UK) stationed in a secondary vented area. All exposure chamber air was exhausted through a series of charcoal traps. The chamber atmosphere was not recycled or returned to the chamber. Sham-exposed control animals were placed in a Dow-modified ADG nose-only chamber of similar design and were provided air of the same quality as exposed rats but not containing SO2F2.
Atmosphere preparation.
The 35SO2F2 was
Chamber concentrations of 30 ppm and 300 ppm were generated by metering at a controlled rate from the stock gas bag into the chamber air stream, where the gas was mixed and diluted to the desired concentration before the air stream entered the chamber. Exposures were extended the appropriate length of time to account for chamber equilibration time (9 min for the 2-liter chamber and 7 min for the 42-liter chamber). The chamber concentrations were monitored with an Agilent Gas Chromatograph 6890A with electron capture detection. The air sample was drawn from the breathing zone of the chamber through a closed gas-sampling loop of 0.53 mm deactivated fused silica capillary line in 300 μl aliquots. Chamber concentrations were measured at least three times during each exposure, in duplicate. The area counts were determined using integration by TurboChrom (PE Nelson, PerkinElmer, Inc, San Jose, CA). A standard curve was generated using known SO2F2 gas standards prior to and after each exposure period. The average integrated area count at each sampling time was determined, the standard curve was applied, and the interpolated values were used to calculate a time-weighted average (TWA) SO2F2 chamber concentration. Non-radiolabled exposures were conducted in a similar manner, except the stock gas bag contained only non-radiolabeled SO2F2.
Blood collection.
The jugular vein–cannulated animals were used only during the radiolabeled portion of the study for blood collection during and after exposure. All other specimens were collected from non-cannulated animals. Certain tissues, noted below, and feces were solubilized with Soluene 350 tissue solubilizer (PerkinElmer, Inc.) according to the methods described by the manufacturer. For the radiolabeled portion of the study, four jugular vein–cannulated rats and four non-cannulated rats were exposed to each 35SO2F2 concentration. Venous blood samples (0.15 ml) were collected via the indwelling jugular cannulae at 0.25, 0.5, 1, 2, 3, and 4 h during the inhalation exposure and at 0.5, 1, 2, 4, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post-exposure. Blood sampling times are designated in the text and figures as –3.75, –3.5, –3, –2, –1, and 0 h for the 0.25, 0.5, 1, 2, 3, and 4 h during-exposure samples, respectively. Blood was obtained at sacrifice via cardiac puncture. The blood was centrifuged to separate plasma and red blood cells (RBC). Plasma and RBC were analyzed for radioactivity from which plasma and RBC 35S concentration-time courses were constructed. Aliquots of plasma were counted directly by LSS while RBC were solubilized and then analyzed by LSS. In addition, approximately equal-volume aliquots of blood were pooled (per sample time) and stored at –80 °C until chemical analysis.
During the non-radiolabeled portion of the study, groups of three rats were sacrificed at –2 h (during exposure), at 0 h (end of exposure), and at 2, 4, 8, and 20 h post-exposure. Sham-exposed control animals in groups of two were sacrificed at –4, 0, 4, 8 and 20 h. Blood was obtained at sacrifice via cardiac puncture. The blood was centrifuged to separate plasma and RBC, and the plasma was analyzed by fluoride ion–specific probe (see below) for fluoride ion content, and a fluoride ion concentration-time course was constructed. Plasma was not pooled for fluoride analysis.
Urine specimens.
For non-cannulated rats exposed to 35SO2F2, all urine voided during the study was collected in dry-ice–cooled traps. The urine traps were changed at the end of exposure (0 h) and at 6, 12, 24, 48, 72, 96, 120, 144, and 168 h post-exposure. Each urine specimen and rinse was analyzed for radioactivity by LSS as described below. Equal volume aliquots of urine samples from the 0–6 h and 6–12 h post-exposure collection intervals were pooled and stored at –80°C prior to radiochemical profile analysis.
Feces specimens.
For non-cannulated rats exposed to 35SO2F2, all feces were collected in dry-ice–chilled containers through the exposure period (0 h) and at 24, 48, 72, 96, 120, 144, and 168 h post-exposure. Aqueous homogenates (25% w/w) were prepared, and weighed aliquots of these homogenates were solubilized and quantitated for radioactivity by LSS. In addition, equal volume aliquots of fecal homogenates were taken from the 0–24 h post-exposure collection interval and pooled. These pooled samples were stored at –80°C prior to radiochemical profile analysis.
Expired air.
Air was drawn through the metabolism cage at approximately 500 ml/min. Where expired air exited the cage, volatiles in it were trapped on charcoal by passage of the expired air through a charcoal trap. The charcoal was then desorbed with toluene and aliquots were analyzed for radioactivity by LSS. No radioactivity was detected in the 0–24 h post-dosing charcoal traps after the 300 ppm exposure. Therefore, expired air was not passed through charcoal for the remainder of the 300 ppm sample collection intervals nor for any of the sample collection intervals for the 30 ppm exposure.
Terminal sacrifice.
At 168 h post-exposure, the animals were anesthetized with CO2 and killed by exsanguination via cardiac puncture. The metabolism cages were then washed, and the cage wash was analyzed for radioactivity.
Tissues.
The following tissues were collected at sacrifice: terminal blood, brain, gastrointestinal (GI) tract, kidney, liver, lung, perirenal fat, residual carcass, respiratory turbinate, olfactory turbinate, skin, and spleen. The respiratory and olfactory turbinates were surgically separated and removed during dissection. The brain, GI tract plus contents, kidney, liver, lung, carcass, and spleen were collected, homogenized individually (33% homogenate), and a weighed aliquot was solubilized and analyzed for radioactivity by LSS. Blood was obtained at sacrifice via cardiac puncture, and an aliquot was solubilized and analyzed for radioactivity by LSS. The skin was removed from the carcass and a representative skin sample was solubilized and analyzed for radioactivity by LSS. Perirenal fat and the nasal turbinates, both olfactory and respiratory, were directly solubilized without homogenization.
For the non-radiolabeled fluoride ion portion of the study, aqueous homogenates of brain and kidney from groups of three rats sacrificed at –2 h (during exposure), 0 h (end of exposure), 2 h, and 4 h post-exposure were analyzed for fluoride ion by fluoride ion–specific probe as described below.
35S-Analysis.
Radioactivity was quantified in a liquid scintillation spectrometer (Beckman LS-1801, LS-3801, or LS-6000 or Packard Tri Carb 2900TR). Samples were counted within 24 h after collection; therefore no correction for radioactive decay was performed (half-life 35S = 88 days).
Chemical analysis.
Whole blood samples submitted for radiochemical profiling were collected directly into tared glass vials containing 0.5 ml of acetonitrile extraction solvent. These vials were sealed, vortex mixed 30 s, and centrifuged at 3000 rpm for 10 min, after which the liquid was transferred to 1.5-ml autosampler vials for high pressure liquid chromatography/radioactivity monitor (HPLC/RAM) analysis. Because of the low levels of radioactivity in the samples, 20-s fractions were collected and assayed by LSS to improve the method detection limit.
Urine samples for radiochemical analysis were selected based upon the amount of radioactivity present in the samples. Pooled urine samples from the –4 to 0 h exposure period interval and the 0 to 6 h, 6 to 12 h, and 12 to 24 h (300 ppm only) post-exposure intervals were analyzed. The urine samples were pooled by combining equal 250 μl aliquots from each of the four animals per collection interval. The vials were centrifuged at 3000 rpm for 15 min, and the liquid was transferred to 1.5-ml autosampler vials for HPLC/RAM analysis. Because of the low levels of radioactivity in the samples, fractions were collected and assayed by LSS to improve the method detection limit.
The LSS data were used to generate reconstructed radiochromatograms, and these radiochromatograms were integrated to determine peak area response of the radiolabeled peaks. The concentrations of sulfate and fluorosulfate in these samples were estimated from the calculated specific activity of sulfate and fluorosulfate and the total radioactivity in the radiolabeled peaks. The concentration of fluoride ion in the urine of rats exposed to both concentrations of 35SO2F2 was determined by ion selective electrode (ISE; Orion Research, Cambridge, MA). Also, ISE determination of the fluoride ion concentration in the plasma, as well as the brain and kidney tissue homogenates, of rats exposed to both concentrations of non-radiolabeled SO2F2 was conducted.
Data analysis.
Radioactivity data are presented as mean ± SD of four animals, and ISE tissue fluoride data are presented as mean ± SD of three animals. All other data are single measurements of samples pooled per exposure concentration and time. Radioactivity was converted to SO2F2 concentration by dividing the measured radioactivity value by the specific activity of the SO2F2 gas in the chamber, and the results are expressed as microgram or micromole equivalents (μg-Eq or μmol-Eq) SO2F2. Pharmacokinetic analyses were performed on individual plasma and RBC concentration-time data to calculate the area under the concentration-time curves (AUC) and half-life of elimination of 35S using PK Solutions pharmacokinetic modeling program (Summit Research Services, Montrose, CO). Fluoride levels in the plasma, brain, and kidney were evaluated by analysis of variance (ANOVA). The analyses were carried out at alpha = 0.05. Plasma fluoride statistical analysis was conducted with 0, 4, 8, and 20 h samples. Brain and kidney fluoride statistical analysis was conducted with 0 and 4 h samples.
RESULTS
Sulfuryl Fluoride Exposure
The nominal exposure concentration of 300 ppm 35SO2F2 resulted in an actual time-weighted average (TWA) of 274 ppm through the 4-h exposure period and a 35S concentration of approximately 2.8 μCi/l of atmosphere (specific activity 0.25 mCi/mmol). The nominal non-radiolabeled 300 ppm SO2F2 exposure resulted in a TWA of 312 ppm. The nominal exposure concentration of 30 ppm 35SO2F2 resulted in an actual TWA of 28.4 ppm through the 4-h exposure period and a 35S concentration of approximately 0.26 μCi/l of atmosphere (specific activity 0.22 mCi/mmol). The nominal non-radiolabeled 30 ppm SO2F2 exposure resulted in a TWA of 31.2 ppm. SO2F2 was not detected in any of the analyses of the control chamber atmosphere at a level exceeding the lowest level quantified [1 ppm].
All animals survived the 4-h SO2F2 exposure. Those animals exposed to 30 or 300 ppm 35SO2F2 were maintained for 1 week post-exposure prior to sacrifice. Animals exposed to non-radiolabeled SO2F2 were sacrificed at the specified times pre-, during, and post-exposure.
Plasma Radioactivity
Radioactivity derived from 35SO2F2 achieved quantifiable plasma levels by 15 to 30 min after initiation of the exposure (Fig. 1). Plasma levels of radioactivity continuously increased throughout the exposure period, ultimately achieving peak mean plasma concentrations of 5.2 and 38 μg-Eq SO2F2/g at the end of the 30 and 300 ppm exposures, respectively. After termination of 35SO2F2 exposure the plasma radioactivity decreased rapidly during an initial phase with a half-life of 2.5 h for both exposure concentrations. A secondary phase beginning about 24 h post-exposure was observed with a half-lives of 83 and 56 h for the 30 and 300 ppm exposures, respectively. The plasma AUC(–4 h to ) for the 30 and 300 ppm exposures were calculated as 96.7 and 756 μg-h ml–1.
RBC Radioactivity
The kinetics of RBC-associated radioactivity were similar to that of plasma radioactivity in uptake and initial distribution phases, but a longer terminal half-life for the elimination of RBC radioactivity was observed (Fig. 2). Peak RBC radioactivity was measured at the end of the 4-h exposure period for both 30 and 300 ppm 35SO2F2 exposures. Immediately following the 35SO2F2 exposures, mean peak RBC radioactivity reached 4.7 and 40.3 μg-Eq SO2F2/g RBC for the 30 and 300 ppm 35SO2F2 exposure concentrations, respectively. After the peak RBC radioactivity levels at termination of exposure, an initial elimination phase half-life of 2.5 and 1.1 h was calculated for the 30 and 300 ppm exposure levels, respectively. After the 30 ppm exposure the phase was estimated to be 222 h and following the 300 ppm exposure the phase was estimated at 139 h. These longer terminal half-lives may be a result of either (1) nonspecific incorporation of the 35S radiolabel as the radiolabel becomes available through normal sulfate pool metabolism, transformed into amino acids, and incorporated into tissues or (2) a result of binding of some unknown 35S-containing component to RBC. Radioactivity remained at measurable, albeit small, concentrations until the time of terminal sacrifice, 168 h post-exposure. The AUC(–4 h-) values for RBC, 863 and 5492 μg-h ml–1 for 30 and 300 ppm exposure concentrations, respectively, are 9-fold to 7-fold larger than the AUC obtained for plasma.
Excretion
Once absorbed, the 35SO2F2-derived radioactivity rapidly appeared in the urine and feces (Fig. 3). Urine contained 88.9% and 85.6% of the total excreted radioactivity through 7 days post-exposure for the 30 and 300 ppm SO2F2 exposures, respectively. The urine samples excreted during the exposure period contained 273 and 2766 μg-Eq SO2F2, or 42% and 51% of the total urinary radioactivity, for the 30 and 300 ppm SO2F2 exposures, respectively. The 0 to 6 h interval urine samples collected immediately following the end of the exposures contained 167 and 936 μg-Eq SO2F2, or an additional 26% and 17% of the total urinary radioactivity, for the 30 and 300 ppm SO2F2 exposures, respectively. Initial urinary elimination rates of 68 and 691 μg-Eq h–1 for 30 and 300 ppm exposures, respectively, rapidly decreased through subsequent collection intervals. An initial urinary half-life for 35SO2F2-derived radioactivity was estimated as approximately 4 h at both exposure concentrations. This was followed by a second urinary elimination phase with a half-life of approximately 40 h. Radioactivity remained detectable in the urine through 7 days post-exposure.
Some radioactivity was detected in feces collected during the exposure period, probably from contamination of fecal pellets with radioactive urine. Separation and collection of urine and feces during the nose-only exposure period was difficult because of the animal restraint device used with the nose-only exposure chamber. After the exposures, when cleaner separation of urine and feces was possible, less than 10% of the total excreted radioactivity was collected with the feces. Through 48 h post-exposure, 70 and 704 μg-Eq SO2F2 was collected in the feces from the 30 and 300 ppm exposures, respectively, representing 91–93% of the total amount of radioactivity recovered in the feces. In total, through 7 days post-dosing, 73 and 777 μg-Eq SO2F2 were recovered in the feces from the 30 and 300 ppm exposure groups, respectively.
Tissue Distribution
The disposition of radioactivity in selected tissues 7 days after exposure to 30 or 300 ppm 35SO2F2 is presented in Figure 4. After both the 30 and 300 ppm exposures, the lungs had the highest concentration of radioactivity, 0.77 and 6.30 μg-Eq SO2F2/g tissue, respectively. The rank order for the concentration of radioactivity at 30 ppm was lung with the highest concentration followed by spleen, kidneys, nasal turbinates (respiratory and olfactory), brain, skin, carcass, liver, GI tract, and fat. The rank-order following the 300 ppm exposure was similar to that obtained with the 30 ppm exposure, except that the turbinates followed the lungs in the concentration of radioactivity in the tissues. In general, the concentrations of radioactivity in the tissues were 7- to 11-fold higher after the 300 ppm exposure than after the 30 ppm exposure.
Total Absorption
Summation of the recovered radioactivity from urine, feces, and tissues gives an approximation of the total absorbed radioactivity. The total absorbed radioactivity can be used as an estimate of the amount of SO2F2 that became systemically available during the 4-h exposures. After the 30 ppm exposure, 581, 73, and 35 μg-Eq SO2F2 (5.7, 0.7, and 0.34 μmol-Eq) were recovered in the urine, feces, and tissues, respectively. Therefore, about 688 μg-Eq SO2F2 (6.7 μmol-Eq) were absorbed during the 4-h 30 ppm exposure, and 85% was eliminated in the urine. After the 300 ppm exposure, 4618, 777, and 298 μg-Eq SO2F2 (45, 7.6, and 2.9 μmol-Eq) were recovered in the urine, feces, and tissues, respectively. Therefore, about 5690 μg-Eq SO2F2 (55 μmol-Eq) were absorbed during the 4-h 300 ppm exposure and 81% was eliminated in the urine. The 5% of the 35S radiolabel recovered from tissues 7 days post-exposure is likely left after non-specific incorporation of the 35S radiolabel, as absorbed radiolabeled sulfur compounds are transformed into amino acids and become incorporated into tissues (Strandberg, 1964).
Chemical Analysis—Blood
Whole blood samples, selected for analysis based on the amount of radioactivity present, were extracted with acetonitrile and analyzed by HPLC/RAM via an ion chromatographic separator. Conventional ion chromatography employs a fixed ion strength mobile phase with suppressed conductivity detection. However, this technique produced poor peak shapes for sulfate and fluorosulfate, which substantially lowered the detection limits. Therefore, the samples in this study were separated with an ion strength gradient and RAM detection. This gradient performed well with acceptable separation of two metabolites and good peak shape (Fig. 5).
Only two radiolabeled components were present in the blood extract samples. A 35S-sodium sulfate standard (Amersham Biosciences, Piscataway, NJ) was used to identify the first metabolite peak in the urine and blood profiles as 35S-sulfate by co-chromatography. The identification of the second peak as fluorosulfate was confirmed by 19F NMR spectroscopy.
Although not directly assayed, there was no evidence of parent 35S-sulfuryl fluoride in the blood based on radiochemical profiles. The presence of SO2F2 in blood was not expected, as work previously done in this laboratory showed, in vitro, rapid removal of SO2F2 from rat blood fortified with high levels of SO2F2 (t < 3 min) (unpublished data, The Dow Chemical Company).
A whole blood time-course of sulfate and fluorosulfate is presented in Figure 6. The amount of fluorosulfate is approximately 2-fold higher than sulfate at all sample times, except at 15 min after the beginning of the 300 ppm exposure, when the concentration of fluorosulfate is 6.5-fold higher, and 4 h after the end of the 300 ppm SO2F2 exposure, when only a small amount of fluorosulfate was detected. At all sample times, sulfate and fluorosulfate are approximately 3- to 5-fold higher after the 300 ppm exposure than after the 30 ppm exposure. Based on the limited amount of data available, a half-life for fluorosulfate elimination from whole blood was calculated to be 48 to 73 min, whereas the half-life for sulfate elimination from whole blood was calculated to be 50 to 64 min.
Chemical Analysis—Urine
Pooled urine samples, with selection and analysis of samples based on the amount of radioactivity present, were analyzed by HPLC/RAM. Two radioactive peaks tentatively identified as sulfate (by co-chromatography with 35S-sulfate) and fluorosulfate (by 19F NMR spectroscopy) were detected in urine (Fig. 5). The presence of SO2F2 in urine was not expected, as SO2F2 has been shown to be rapidly hydrolyzed in aqueous solutions (t < 18 min at pH = 8.07; Cady and Misra, 1974). During the exposure period, the amount of fluorosulfate eliminated in the urine was 3- to 3.5-fold higher than the amount of sulfate (Fig. 7). After the exposures, the amount of sulfate recovered in the urine was greater than the amount of fluorosulfate recovered in urine. Six to twelve h post-exposure, 5- to 7-fold more sulfate was recovered in the urine than fluorosulfate. The total μmoles of sulfate plus fluorosulfate recovered in the urine as determined by HPLC/RAM (3.8 and 43 μmoles; 30 and 300 ppm, respectively) compares well with the μmol-Eq SO2F2 in the urine + rinse as determined from the radiolabeled portion of this study (5.7 and 45 μmoles; 30 and 300 ppm, respectively).
Conversion of sulfate and fluorosulfate urine concentrations to rate estimates allowed calculation of half-lives for the elimination of sulfate and fluorosulfate in urine. Sulfate was eliminated in the urine with a half-life of 2.2 and 3.8 h for the 30 and 300 ppm exposure groups, respectively. Fluorosulfate was eliminated slightly faster, with a half-life estimate of 1.2 and 2.4 h for the 30 and 300 ppm exposure groups, respectively.
Fluoride Analysis
The ISE technique used in this study measures free fluoride only. Three types of fluoride make up the "total" fluoride level in tissues, ionic (free), acid labile, and nonionic (bound) (for a review, see Singer and Ophaug, 1982). Acid labile fluoride accounts for a very small amount of the total fluoride (0.01 ppm). However, the contribution of nonionic (bound) fluoride to the total fluoride level can be as high as 50% of the total in plasma. The determination of nonionic fluoride involves a very labor-intensive combustion method that provides highly variable results (ibid.), and therefore was not included in this study.
Ion selective electrode analysis of plasma and tissues was conducted with rats exposed to non-radiolabeled SO2F2 and with control rats exposed to clean air. Levels of plasma fluoride ion in control animals ranged from 0.023 to 0.070 μmol/ml plasma through a 24-h cycle. These measurements are within the control range of serum fluoride concentration reported by Eisenbrandt and Nitschke (1989).
An apparent slight elevation of plasma fluoride from control levels was observed during the 30 ppm and 300 ppm exposures, achieving concentrations of 0.046 and 0.132 μmol/ml plasma, respectively. But, plasma fluoride levels rapidly returned to control levels (0.03–0.05 μmol/ml plasma) by about 2 h after exposure was terminated (Fig. 6). Plasma fluoride was 1.6- and 5.4-fold higher than control levels at the end of exposure for the 30 and 300 ppm SO2F2 exposures, respectively. The maximum concentration of plasma fluoride measured at the termination of the 4-h 30 ppm SO2F2 exposure was 0.04 μmol/ml and is similar to that reported by Eisenbrandt and Nitschke (1989) following 6-h/day, 5 days/week for 13 weeks exposure to 30 ppm SO2F2, although considerable variation was reported. The plasma concentration reported here after the 300 ppm exposure was 0.13 μmol/ml and is nearly twice as large as that reported by Eisenbrandt and Nitschke (1989). However, our data indicate a rapid clearance of fluoride from the plasma and a return to background levels within 2 h following termination of exposure. Because serum was collected from the rats in the 13-week study at necropsy the day after the final exposure, measurement of serum fluoride was conducted subsequent to the time of peak levels of fluoride. Figure 6 presents the blood sulfate plus fluorosulfate data and the plasma fluoride data together in one figure for each exposure concentration. The overall shapes of the curves during and after the SO2F2 exposures are quite similar, indicating that the kinetics of formation and elimination from plasma of these three metabolites are similar or are interrelated.
Fluoride ion analysis of urine from non-exposed rats and from rats exposed to SO2F2 was conducted with selected urine samples. Figure 7 presents the urine sulfate plus fluorosulfate data and the fluoride data together in one figure for each exposure concentration. As observed with plasma, the overall shapes of the curves during and after the SO2F2 exposures are quite similar, indicating that the kinetics of formation and elimination of these three metabolites are similar or are interrelated.
Elevated levels of fluoride ion were detected in urine during and after the SO2F2 exposures. Non-exposed control rats had concentrations of fluoride ion of approximately 0.12–0.13 μmol/ml urine. By the end of the 4-h 30 ppm exposure, the urine levels of fluoride ion reached a maximum concentration of 0.5 μmol/ml urine. This concentration was maintained through 6 h post-exposure. By 12 h post-exposure the concentration had diminished to near background levels, 0.14 μmol/ml urine. In a similar fashion, by the end of the 4-h 300 ppm exposure the urine levels of fluoride ion reached a maximum concentration of 4.0 μmol/ml urine, about 8-fold higher than that obtained after the 30 ppm exposure. However, this concentration diminished through 6 h post-exposure to 1.7 μmol/ml urine and by 24 h post-exposure to 0.3 μmol/ml urine.
Fluoride levels in brain and kidney tissue during and after exposure to 30 and 300 ppm SO2F2 are presented in Figure 8. Kidney fluoride levels were roughly 2- to 2.5-fold higher at all collection times than those measured in control rats. Control rats had mean fluoride levels of 0.12 μmol/g kidney tissue, while both 30 and 300 ppm exposure levels resulted in mean fluoride concentrations of about 0.26 μmol/g kidney tissue. These levels were measured by the second hour of exposure and were maintained through 4 h post-exposure.
A slight 1.5-fold elevation in fluoride levels in brain tissue relative to control rats was observed during and after exposure to 30 ppm SO2F2. The mean control brain tissue fluoride ion concentrations were roughly 0.03 μmol/g tissue at all sacrifice times. Fluoride levels increased to 0.04 μmol/g brain after the 30 ppm SO2F2 exposure, whereas after the 300 ppm SO2F2 exposure, concentrations as high as 0.12 μmol/g tissue were measured, 4-fold higher than controls, at the end of the exposure period. The concentrations of fluoride in brain tissue approached the control levels by 4 h post-exposure.
DISCUSSION
Radiolabeled SO2F2 was rapidly absorbed via inhalation exposure, achieving maximum concentrations of 35SO2F2-derived radioactivity in both plasma and red blood cells near the end of the 4-h exposure periods. Saturation of absorption was not achieved during these 4-h exposures as evidenced by the following findings: (1) the measured levels of radioactivity were roughly proportional to the exposure concentrations, (2) the level of plasma/RBC radioactivity was increasing at the end of exposure, (3) steady-state 35S conditions were not achieved, and (4) the initial half-lives for clearance of radioactivity from plasma and RBC were the same at both exposure concentrations. Plasma levels of radioactivity during the 4-h exposure never reached steady-state conditions, most likely because of rapid elimination of radioactivity from the plasma. The similar peak concentrations of radioactivity measured with plasma and RBC suggests the 35S initially was evenly distributed between these two compartments. Once absorbed, the 35S was rapidly excreted, primarily via the urine. A large portion of absorbed 35S was excreted in the urine, even during the 4-h nose-only exposure.
Seven days post-exposure, small amounts of the radiolabel were evenly distributed among the tissues, suggesting incorporation into the tissues of 35S that had been transformed into amino acids; however, the chemical composition was not determined. Radioactivity was recovered mainly in respiratory tissues, which are the site of first exposure to the gas. The lungs had the highest levels of radioactivity 7 days post-exposure, and the nasal turbinates also had detectable radioactivity. Radioactivity associated with the RBC remained elevated 7 days post-exposure. Highly perfused tissues such as spleen and kidney also had higher levels of radioactivity than non-respiratory tissues, a finding attributed to the radioactivity in the blood. Radioactivity was rapidly cleared from plasma and RBC with initial half-lives of 2.5 h after the 30 ppm exposure and 1–2.5 h after the 300 ppm exposure. However, the terminal half-life of radioactivity was 2.5-fold longer in RBC than in plasma. The identification of fluorosulfate and sulfate in blood and urine suggests that SO2F2 is first hydrolyzed to fluorosulfate, with release of fluoride, followed by further hydrolysis to sulfate and release of the remaining fluoride. This apparent metabolism is supported by the increases in fluoride detected in the blood and urine following exposure of rats to SO2F2.
Fluoride metabolism resulting from inhalation exposure to SO2F2 would be expected to be consistent with the metabolism of fluoride from other sources. Whitford (1996) provides a comprehensive review of the metabolism of fluoride. Once absorbed, plasma functions as the "central compartment" for fluoride. Fluoride rapidly establishes a steady-state distribution between extracellular and intracellular fluids, and thus fluoride levels in these two compartments parallel each other.
The major route of fluoride elimination from the body is excretion in the urine. The kidneys are very efficient in removing fluoride from the body. The concentration of ionic fluoride in the glomerular filtrate is very similar to that of plasma, and there is no evidence for the tubular secretion of fluoride, although 62–78% of the filtered fluoride is reabsorbed. Approximately 50% of an absorbed amount of fluoride is excreted in the urine within 24 h, whereas most of the remainder becomes associated with calcified tissue.
Fluoride associated with calcified tissues is not irreversibly bound, and this is especially true of recently acquired fluoride on the surfaces of bone crystallites. If the intake of fluoride were to increase or decrease on a chronic basis, the concentrations in the calcified tissues would eventually reflect the change. Fluoride accumulation by bone is inversely related to age. The ability of the developing skeleton to clear fluoride from plasma more rapidly than the mature skeleton is largely a function of surface area (mature bone is compact, whereas the crystallites of developing bone provide extensive surface are for reactions involving fluoride). However, data in rats indicate that the uptake of fluoride by the skeleton continues throughout the first 18 months of life (75–85% of life span) and accounts for one half the plasma clearance during this period.
A likely cause of SO2F2 toxicity is the metabolic release of fluoride ions as postulated by Nitschke, et al. (1986) and Nitschke and Eisenbrandt (2001). The data presented here support the hypothesis that SO2F2 toxicity is the result of metabolic release of fluoride ions. Inhaled SO2F2 is rapidly absorbed and hydrolyzed to fluorosulfate and ionic fluoride, followed by further hydrolysis to sulfate and an additional fluoride ion. Therefore, these data suggest that the toxicity elicited by SO2F2 may be due to the release of fluoride ions, rather than a direct toxic action of SO2F2.
NOTES
The studies reported in this article were presented at the 42nd annual meeting of the Society of Toxicology, March 2003, Salt Lake City, UT (The Toxicologist 72, 713, March 2003).
VIKANE is a trademark of the Dow AcroSciences LLC.
ACKNOWLEDGMENTS
The authors thank T. Card, A. J. Clark, J. Y. Domoradzki, M. J. Filary, J. A. Hotchkiss, C. E. Houtman, M. A. Knoerr, S. M. Krieger, G. T. Marty, L. G. McFadden, E. S. Mullen, D. L. Rick, S. Saghir, and C. M. Thornton for their excellent technical assistance. Conflict of interest: The Dow Chemical Company, Dow AgroSciences LLC.
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Nitschke, K. D., and Eisenbrandt, D. L. (2001). Sulfuryl fluoride. In Handbook of Pesticide Toxicology, Agents, 2nd Edition, Chap. 88, Vol 2. (Robert Krieger, ed.), Academic Press, San Diego, CA.
Nitschke, K. D., Albee, R. R., Mattsson, J. L., and Miller, R. R. (1986). Incapacitation and treatment of rats exposed to a lethal dose of sulfuryl fluoride. Fundam. Appl. Toxicol. 7, 664–670.
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Singer, L., and Ophaug, R. (1982). Ionic and nonionic fluoride in plasma (or serum). Crit. Rev. Clin. Lab. Sci. 18, 111–140.
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Dow AgroSciences LLC, Indianapolis, Indiana 46268
ABSTRACT
Sulfuryl fluoride (SO2F2) is a structural fumigant gas used to control drywood termites and wood-boring beetles. The pharmacokinetics and metabolism of inhaled SO2F2 were evaluated in male Fischer-344 rats exposed to 30 or 300 ppm 35S-labeled SO2F2 for 4 h. Blood, urine and feces were collected during and after the exposures and analyzed for radioactivity, 35S-labeled fluorosulfate and sulfate, and fluoride (urine and feces only). Selected tissues were collected 7 days post-exposure and analyzed for radioactivity. During and after unlabeled SO2F2 exposures, blood, brain, and kidney were collected and analyzed for fluoride ion. SO2F2 was rapidly absorbed, achieving maximum concentrations of radioactivity in both plasma and red blood cells (RBC) near the end of the 4-h exposure period. Radioactivity was rapidly excreted, mostly via the urine. Seven days post-exposure, small amounts of radioactivity were distributed among several tissues, with the highest concentration detected in respiratory tissues. Radioactivity associated with the RBC remained elevated 7 days post-exposure, and highly perfused tissues had higher levels of radioactivity than other non-respiratory tissues. Radioactivity cleared from plasma and RBC with initial half-lives of 2.5 h after 30 ppm and 1–2.5 h after 300 ppm exposures. The terminal half-life of radioactivity was 2.5-fold longer in RBC than plasma. Based on the radiochemical profiles, there was no evidence of parent 35SO2F2 in blood. Identification of fluorosulfate and sulfate in blood and urine suggests that SO2F2 is hydrolyzed to fluorosulfate, with release of fluoride, followed by further hydrolysis to sulfate and release of the remaining fluoride.
Key Words: sulfuryl fluoride; fluorosulfate; fluoride; inhalation; rat.
INTRODUCTION
Sulfuryl fluoride (SO2F2, VIKANE gas fumigant) is a colorless, odorless gas that has been used to fumigate buildings since 1961 to control pests, including drywood termites and wood-boring beetles. The American Conference of Governmental Industrial Hygienists (ACGIH) threshold limit value and OSHA permissible exposure level for SO2F2 are 5 ppm TWA and 10 ppm STEL (ACGIH, 1998). Extensive toxicological studies have been conducted with SO2F2 over the past 30 years, and the toxicity is well characterized (reviewed in Nitschke and Eisenbrandt, 2001). SO2F2 is acutely toxic to mammals when inhaled, with a LC50 (4 h) for rats of about 1000 ppm. Rats exposed to SO2F2 at concentrations of 1500 ppm or 2000 ppm showed central nervous system depression (sedation) within 20 to 40 min, and continued exposure resulted in convulsions and adverse effects on the respiratory tract, liver, and kidneys, followed by death within 3–4 h (unpublished data of The Dow Chemical Company). Exposure of rats to 4000, 10,000, 20,000, or 40,000 ppm SO2F2 decreased the time to incapacitation and death as the vapor concentration increased (Nitschke et al., 1986).
Fluoride ion may play a role in the mechanism of action of SO2F2 in insects and mammals. Many of the observations in rodents overexposed to SO2F2 are typical of acute fluoride poisoning (Drill, 1954; Goodman et al., 1980; Greenwood, 1940; Whitford, 1996). Eisenbrandt and Nitschke (1989) studied the toxicity of SO2F2 in rats and rabbits exposed to 0, 30, 100, or 300 ppm for 6 h/day, 5 days/week for 13 weeks. A statistically significant and dose-related increase in ionic fluoride concentration was reported in the serum of rabbits exposed to SO2F2 for 13 weeks, although serum levels of ionic fluoride were not significantly elevated in rats. Inhalation exposure of rats to 300 ppm SO2F2 resulted in decreased body weights; histopathological changes in the respiratory tract, brain, and kidneys; as well as mottled teeth and decreased specific gravity of the urine. At 100 ppm, the tooth enamel changes were the only exposure-related changes in the rats. No treatment-related effects were observed at 30 ppm. Neuropathological changes in the rats consisted of minimal vacuolation in the area of the caudate-putamen nuclei that was more prominent in the white fiber tracts of the internal capsule than in the adjacent neuropil. Alteration of neurological function in the rats exposed to 300 ppm SO2F2 for 13 weeks was demonstrated by a decreased flicker fusion threshold, and a slowing of flash, auditory, and somatosensory evoked potentials (Mattsson et al., 1988). The functional responses as well as brain histology were within normal limits at 2 months post-exposure.
Mottled incisor teeth in rats exposed to 100 or 300 ppm SO2F2 for 13 weeks were consistent with an increase in systemic fluoride as produced by several other inorganic and organic fluoride compounds (Eagers, 1969; Pattison, 1959). The current metabolism and disposition study was undertaken to better understand the mammalian toxicity of SO2F2 and to determine the systemic availability of fluoride following SO2F2 exposure.
MATERIALS AND METHODS
Test material.
Sulfuryl fluoride, radiolabeled with 35sulfur, was obtained from Moravek Biochemicals, Inc., Brea, California with a specific activity of 10 mCi/mmol. A radiochemical purity of 100% 35SO2F2 was established using gas chromatography with flow radiogas monitoring and identification by retention time confirmation using an authentic standard of SO2F2 and thermal conductivity detection. Structural confirmation was accomplished by gas chromatography/electron impact ionization/mass spectrometry. Non-radiolabeled SO2F2 was obtained from Dow AgroSciences LLC (Indianapolis, IN). Analysis of this test material prior to the study revealed the sample to be 99.8% SO2F2. All other chemicals and solvents were reagent grade or better.
Animals.
Male Fischer-344 rats (197–242 g) were used because they were the same strain used in previous inhalation toxicity studies of SO2F2 (Eisenbrandt and Nitschke, 1989; Mattsson et al., 1988; Nitschke et al., 1986). Upon arrival at the laboratory, which is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International), each animal was evaluated by a laboratory veterinarian and found to be in good health and acceptable for study purposes. The rooms in which the animals were housed had the relative humidity maintained within a range of 40–70% and the room temperature maintained at 22° ± 3°C. An approximate 12 h light/dark photocycle was maintained for all animal rooms. Room air was exchanged 12–15 times/h. On each of 2 days prior to exposure, the animals were stage-acclimated to the nose-only chamber (1 h on day 1, 2 h on day 2), with the nose cones connected to the exposure chamber and airflow comparable to what it was during exposure.
Rats cannulated in the jugular vein by the supplier (Hilltop Lab Animals, Inc. Scottsdale, PA) were used for the 35S time-course experiments. To increase the likelihood that the cannulae remained patent, acclimation of the rats to the laboratory environment was limited to 4 days prior to 35SO2F2 exposure. They were returned to their cages for 24 h before 35SO2F2 exposure, and during this period the rats underwent exteriorization of the cannulae under light isoflurane anesthesia, approximately 18 h prior to 35SO2F2 exposure.
Non-cannulated rats used for the remainder of the study were obtained from Charles River Laboratories (Raleigh, NC). The animals used for 35S disposition were acclimated to metabolism cages for at least 2 days. They were returned to the metabolism cages for 24 h before 35SO2F2 exposure. Non-cannulated animals used for fluoride analysis were housed one per cage in stainless-steel cages. Non-cannulated animals were selected from those available and assigned to exposure groups using a computer-driven randomization procedure.
Following exposure to 35SO2F2, non-cannulated animals were housed one per cage in glass metabolism cages. Air was drawn through the metabolism cages at 500 ml/min. Cannulated animals were housed in plastic tubs with corncob bedding for the duration of blood collection. Animals exposed to non-radiolabeled SO2F2 were housed one per cage in stainless-steel cages containing a feed container and a pressure activated nipple-type watering system. Cages were suspended above catch pans, and feed and water were provided ad libitum except during the exposures. Animals were provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, MO) in pelleted form. To minimize the animals' exposure to environmental fluoride, the animals were given Milli-Q de-ionized water (Millipore Co., Bedford, MA) after their arrival at the laboratory. No attempt was made to remove/reduce fluoride ion from nonaqueous sources, i.e., feed (13 ppm fluoride ion, Nutritional Analyses of Certified Rodent Diet #5002 by PMI Nutrition International).
Exposure system.
The 35SO2F2 exposures were conducted in a 2-liter Plexiglas nose-only exposure chamber, stationed in a closed, vented, Plexiglas box, which was located inside a secondary vented area. The rats were held in place by use of a tubular restraint device constructed from #3 mesh galvanized hardware cloth (7.26 mm openings) attached to a polycarbonate nose cone. An excreta separator-collector was attached to the bottom of the restraint device, and the collectors were placed in dry-ice. Chambers used for the non-radiolabeled exposures consisted of a 42-liter Dow-modified ADG nose-only chamber (ADG Developments, Ltd., UK) stationed in a secondary vented area. All exposure chamber air was exhausted through a series of charcoal traps. The chamber atmosphere was not recycled or returned to the chamber. Sham-exposed control animals were placed in a Dow-modified ADG nose-only chamber of similar design and were provided air of the same quality as exposed rats but not containing SO2F2.
Atmosphere preparation.
The 35SO2F2 was
Chamber concentrations of 30 ppm and 300 ppm were generated by metering at a controlled rate from the stock gas bag into the chamber air stream, where the gas was mixed and diluted to the desired concentration before the air stream entered the chamber. Exposures were extended the appropriate length of time to account for chamber equilibration time (9 min for the 2-liter chamber and 7 min for the 42-liter chamber). The chamber concentrations were monitored with an Agilent Gas Chromatograph 6890A with electron capture detection. The air sample was drawn from the breathing zone of the chamber through a closed gas-sampling loop of 0.53 mm deactivated fused silica capillary line in 300 μl aliquots. Chamber concentrations were measured at least three times during each exposure, in duplicate. The area counts were determined using integration by TurboChrom (PE Nelson, PerkinElmer, Inc, San Jose, CA). A standard curve was generated using known SO2F2 gas standards prior to and after each exposure period. The average integrated area count at each sampling time was determined, the standard curve was applied, and the interpolated values were used to calculate a time-weighted average (TWA) SO2F2 chamber concentration. Non-radiolabled exposures were conducted in a similar manner, except the stock gas bag contained only non-radiolabeled SO2F2.
Blood collection.
The jugular vein–cannulated animals were used only during the radiolabeled portion of the study for blood collection during and after exposure. All other specimens were collected from non-cannulated animals. Certain tissues, noted below, and feces were solubilized with Soluene 350 tissue solubilizer (PerkinElmer, Inc.) according to the methods described by the manufacturer. For the radiolabeled portion of the study, four jugular vein–cannulated rats and four non-cannulated rats were exposed to each 35SO2F2 concentration. Venous blood samples (0.15 ml) were collected via the indwelling jugular cannulae at 0.25, 0.5, 1, 2, 3, and 4 h during the inhalation exposure and at 0.5, 1, 2, 4, 6, 12, 24, 36, 48, 72, 96, 120, 144, and 168 h post-exposure. Blood sampling times are designated in the text and figures as –3.75, –3.5, –3, –2, –1, and 0 h for the 0.25, 0.5, 1, 2, 3, and 4 h during-exposure samples, respectively. Blood was obtained at sacrifice via cardiac puncture. The blood was centrifuged to separate plasma and red blood cells (RBC). Plasma and RBC were analyzed for radioactivity from which plasma and RBC 35S concentration-time courses were constructed. Aliquots of plasma were counted directly by LSS while RBC were solubilized and then analyzed by LSS. In addition, approximately equal-volume aliquots of blood were pooled (per sample time) and stored at –80 °C until chemical analysis.
During the non-radiolabeled portion of the study, groups of three rats were sacrificed at –2 h (during exposure), at 0 h (end of exposure), and at 2, 4, 8, and 20 h post-exposure. Sham-exposed control animals in groups of two were sacrificed at –4, 0, 4, 8 and 20 h. Blood was obtained at sacrifice via cardiac puncture. The blood was centrifuged to separate plasma and RBC, and the plasma was analyzed by fluoride ion–specific probe (see below) for fluoride ion content, and a fluoride ion concentration-time course was constructed. Plasma was not pooled for fluoride analysis.
Urine specimens.
For non-cannulated rats exposed to 35SO2F2, all urine voided during the study was collected in dry-ice–cooled traps. The urine traps were changed at the end of exposure (0 h) and at 6, 12, 24, 48, 72, 96, 120, 144, and 168 h post-exposure. Each urine specimen and rinse was analyzed for radioactivity by LSS as described below. Equal volume aliquots of urine samples from the 0–6 h and 6–12 h post-exposure collection intervals were pooled and stored at –80°C prior to radiochemical profile analysis.
Feces specimens.
For non-cannulated rats exposed to 35SO2F2, all feces were collected in dry-ice–chilled containers through the exposure period (0 h) and at 24, 48, 72, 96, 120, 144, and 168 h post-exposure. Aqueous homogenates (25% w/w) were prepared, and weighed aliquots of these homogenates were solubilized and quantitated for radioactivity by LSS. In addition, equal volume aliquots of fecal homogenates were taken from the 0–24 h post-exposure collection interval and pooled. These pooled samples were stored at –80°C prior to radiochemical profile analysis.
Expired air.
Air was drawn through the metabolism cage at approximately 500 ml/min. Where expired air exited the cage, volatiles in it were trapped on charcoal by passage of the expired air through a charcoal trap. The charcoal was then desorbed with toluene and aliquots were analyzed for radioactivity by LSS. No radioactivity was detected in the 0–24 h post-dosing charcoal traps after the 300 ppm exposure. Therefore, expired air was not passed through charcoal for the remainder of the 300 ppm sample collection intervals nor for any of the sample collection intervals for the 30 ppm exposure.
Terminal sacrifice.
At 168 h post-exposure, the animals were anesthetized with CO2 and killed by exsanguination via cardiac puncture. The metabolism cages were then washed, and the cage wash was analyzed for radioactivity.
Tissues.
The following tissues were collected at sacrifice: terminal blood, brain, gastrointestinal (GI) tract, kidney, liver, lung, perirenal fat, residual carcass, respiratory turbinate, olfactory turbinate, skin, and spleen. The respiratory and olfactory turbinates were surgically separated and removed during dissection. The brain, GI tract plus contents, kidney, liver, lung, carcass, and spleen were collected, homogenized individually (33% homogenate), and a weighed aliquot was solubilized and analyzed for radioactivity by LSS. Blood was obtained at sacrifice via cardiac puncture, and an aliquot was solubilized and analyzed for radioactivity by LSS. The skin was removed from the carcass and a representative skin sample was solubilized and analyzed for radioactivity by LSS. Perirenal fat and the nasal turbinates, both olfactory and respiratory, were directly solubilized without homogenization.
For the non-radiolabeled fluoride ion portion of the study, aqueous homogenates of brain and kidney from groups of three rats sacrificed at –2 h (during exposure), 0 h (end of exposure), 2 h, and 4 h post-exposure were analyzed for fluoride ion by fluoride ion–specific probe as described below.
35S-Analysis.
Radioactivity was quantified in a liquid scintillation spectrometer (Beckman LS-1801, LS-3801, or LS-6000 or Packard Tri Carb 2900TR). Samples were counted within 24 h after collection; therefore no correction for radioactive decay was performed (half-life 35S = 88 days).
Chemical analysis.
Whole blood samples submitted for radiochemical profiling were collected directly into tared glass vials containing 0.5 ml of acetonitrile extraction solvent. These vials were sealed, vortex mixed 30 s, and centrifuged at 3000 rpm for 10 min, after which the liquid was transferred to 1.5-ml autosampler vials for high pressure liquid chromatography/radioactivity monitor (HPLC/RAM) analysis. Because of the low levels of radioactivity in the samples, 20-s fractions were collected and assayed by LSS to improve the method detection limit.
Urine samples for radiochemical analysis were selected based upon the amount of radioactivity present in the samples. Pooled urine samples from the –4 to 0 h exposure period interval and the 0 to 6 h, 6 to 12 h, and 12 to 24 h (300 ppm only) post-exposure intervals were analyzed. The urine samples were pooled by combining equal 250 μl aliquots from each of the four animals per collection interval. The vials were centrifuged at 3000 rpm for 15 min, and the liquid was transferred to 1.5-ml autosampler vials for HPLC/RAM analysis. Because of the low levels of radioactivity in the samples, fractions were collected and assayed by LSS to improve the method detection limit.
The LSS data were used to generate reconstructed radiochromatograms, and these radiochromatograms were integrated to determine peak area response of the radiolabeled peaks. The concentrations of sulfate and fluorosulfate in these samples were estimated from the calculated specific activity of sulfate and fluorosulfate and the total radioactivity in the radiolabeled peaks. The concentration of fluoride ion in the urine of rats exposed to both concentrations of 35SO2F2 was determined by ion selective electrode (ISE; Orion Research, Cambridge, MA). Also, ISE determination of the fluoride ion concentration in the plasma, as well as the brain and kidney tissue homogenates, of rats exposed to both concentrations of non-radiolabeled SO2F2 was conducted.
Data analysis.
Radioactivity data are presented as mean ± SD of four animals, and ISE tissue fluoride data are presented as mean ± SD of three animals. All other data are single measurements of samples pooled per exposure concentration and time. Radioactivity was converted to SO2F2 concentration by dividing the measured radioactivity value by the specific activity of the SO2F2 gas in the chamber, and the results are expressed as microgram or micromole equivalents (μg-Eq or μmol-Eq) SO2F2. Pharmacokinetic analyses were performed on individual plasma and RBC concentration-time data to calculate the area under the concentration-time curves (AUC) and half-life of elimination of 35S using PK Solutions pharmacokinetic modeling program (Summit Research Services, Montrose, CO). Fluoride levels in the plasma, brain, and kidney were evaluated by analysis of variance (ANOVA). The analyses were carried out at alpha = 0.05. Plasma fluoride statistical analysis was conducted with 0, 4, 8, and 20 h samples. Brain and kidney fluoride statistical analysis was conducted with 0 and 4 h samples.
RESULTS
Sulfuryl Fluoride Exposure
The nominal exposure concentration of 300 ppm 35SO2F2 resulted in an actual time-weighted average (TWA) of 274 ppm through the 4-h exposure period and a 35S concentration of approximately 2.8 μCi/l of atmosphere (specific activity 0.25 mCi/mmol). The nominal non-radiolabeled 300 ppm SO2F2 exposure resulted in a TWA of 312 ppm. The nominal exposure concentration of 30 ppm 35SO2F2 resulted in an actual TWA of 28.4 ppm through the 4-h exposure period and a 35S concentration of approximately 0.26 μCi/l of atmosphere (specific activity 0.22 mCi/mmol). The nominal non-radiolabeled 30 ppm SO2F2 exposure resulted in a TWA of 31.2 ppm. SO2F2 was not detected in any of the analyses of the control chamber atmosphere at a level exceeding the lowest level quantified [1 ppm].
All animals survived the 4-h SO2F2 exposure. Those animals exposed to 30 or 300 ppm 35SO2F2 were maintained for 1 week post-exposure prior to sacrifice. Animals exposed to non-radiolabeled SO2F2 were sacrificed at the specified times pre-, during, and post-exposure.
Plasma Radioactivity
Radioactivity derived from 35SO2F2 achieved quantifiable plasma levels by 15 to 30 min after initiation of the exposure (Fig. 1). Plasma levels of radioactivity continuously increased throughout the exposure period, ultimately achieving peak mean plasma concentrations of 5.2 and 38 μg-Eq SO2F2/g at the end of the 30 and 300 ppm exposures, respectively. After termination of 35SO2F2 exposure the plasma radioactivity decreased rapidly during an initial phase with a half-life of 2.5 h for both exposure concentrations. A secondary phase beginning about 24 h post-exposure was observed with a half-lives of 83 and 56 h for the 30 and 300 ppm exposures, respectively. The plasma AUC(–4 h to ) for the 30 and 300 ppm exposures were calculated as 96.7 and 756 μg-h ml–1.
RBC Radioactivity
The kinetics of RBC-associated radioactivity were similar to that of plasma radioactivity in uptake and initial distribution phases, but a longer terminal half-life for the elimination of RBC radioactivity was observed (Fig. 2). Peak RBC radioactivity was measured at the end of the 4-h exposure period for both 30 and 300 ppm 35SO2F2 exposures. Immediately following the 35SO2F2 exposures, mean peak RBC radioactivity reached 4.7 and 40.3 μg-Eq SO2F2/g RBC for the 30 and 300 ppm 35SO2F2 exposure concentrations, respectively. After the peak RBC radioactivity levels at termination of exposure, an initial elimination phase half-life of 2.5 and 1.1 h was calculated for the 30 and 300 ppm exposure levels, respectively. After the 30 ppm exposure the phase was estimated to be 222 h and following the 300 ppm exposure the phase was estimated at 139 h. These longer terminal half-lives may be a result of either (1) nonspecific incorporation of the 35S radiolabel as the radiolabel becomes available through normal sulfate pool metabolism, transformed into amino acids, and incorporated into tissues or (2) a result of binding of some unknown 35S-containing component to RBC. Radioactivity remained at measurable, albeit small, concentrations until the time of terminal sacrifice, 168 h post-exposure. The AUC(–4 h-) values for RBC, 863 and 5492 μg-h ml–1 for 30 and 300 ppm exposure concentrations, respectively, are 9-fold to 7-fold larger than the AUC obtained for plasma.
Excretion
Once absorbed, the 35SO2F2-derived radioactivity rapidly appeared in the urine and feces (Fig. 3). Urine contained 88.9% and 85.6% of the total excreted radioactivity through 7 days post-exposure for the 30 and 300 ppm SO2F2 exposures, respectively. The urine samples excreted during the exposure period contained 273 and 2766 μg-Eq SO2F2, or 42% and 51% of the total urinary radioactivity, for the 30 and 300 ppm SO2F2 exposures, respectively. The 0 to 6 h interval urine samples collected immediately following the end of the exposures contained 167 and 936 μg-Eq SO2F2, or an additional 26% and 17% of the total urinary radioactivity, for the 30 and 300 ppm SO2F2 exposures, respectively. Initial urinary elimination rates of 68 and 691 μg-Eq h–1 for 30 and 300 ppm exposures, respectively, rapidly decreased through subsequent collection intervals. An initial urinary half-life for 35SO2F2-derived radioactivity was estimated as approximately 4 h at both exposure concentrations. This was followed by a second urinary elimination phase with a half-life of approximately 40 h. Radioactivity remained detectable in the urine through 7 days post-exposure.
Some radioactivity was detected in feces collected during the exposure period, probably from contamination of fecal pellets with radioactive urine. Separation and collection of urine and feces during the nose-only exposure period was difficult because of the animal restraint device used with the nose-only exposure chamber. After the exposures, when cleaner separation of urine and feces was possible, less than 10% of the total excreted radioactivity was collected with the feces. Through 48 h post-exposure, 70 and 704 μg-Eq SO2F2 was collected in the feces from the 30 and 300 ppm exposures, respectively, representing 91–93% of the total amount of radioactivity recovered in the feces. In total, through 7 days post-dosing, 73 and 777 μg-Eq SO2F2 were recovered in the feces from the 30 and 300 ppm exposure groups, respectively.
Tissue Distribution
The disposition of radioactivity in selected tissues 7 days after exposure to 30 or 300 ppm 35SO2F2 is presented in Figure 4. After both the 30 and 300 ppm exposures, the lungs had the highest concentration of radioactivity, 0.77 and 6.30 μg-Eq SO2F2/g tissue, respectively. The rank order for the concentration of radioactivity at 30 ppm was lung with the highest concentration followed by spleen, kidneys, nasal turbinates (respiratory and olfactory), brain, skin, carcass, liver, GI tract, and fat. The rank-order following the 300 ppm exposure was similar to that obtained with the 30 ppm exposure, except that the turbinates followed the lungs in the concentration of radioactivity in the tissues. In general, the concentrations of radioactivity in the tissues were 7- to 11-fold higher after the 300 ppm exposure than after the 30 ppm exposure.
Total Absorption
Summation of the recovered radioactivity from urine, feces, and tissues gives an approximation of the total absorbed radioactivity. The total absorbed radioactivity can be used as an estimate of the amount of SO2F2 that became systemically available during the 4-h exposures. After the 30 ppm exposure, 581, 73, and 35 μg-Eq SO2F2 (5.7, 0.7, and 0.34 μmol-Eq) were recovered in the urine, feces, and tissues, respectively. Therefore, about 688 μg-Eq SO2F2 (6.7 μmol-Eq) were absorbed during the 4-h 30 ppm exposure, and 85% was eliminated in the urine. After the 300 ppm exposure, 4618, 777, and 298 μg-Eq SO2F2 (45, 7.6, and 2.9 μmol-Eq) were recovered in the urine, feces, and tissues, respectively. Therefore, about 5690 μg-Eq SO2F2 (55 μmol-Eq) were absorbed during the 4-h 300 ppm exposure and 81% was eliminated in the urine. The 5% of the 35S radiolabel recovered from tissues 7 days post-exposure is likely left after non-specific incorporation of the 35S radiolabel, as absorbed radiolabeled sulfur compounds are transformed into amino acids and become incorporated into tissues (Strandberg, 1964).
Chemical Analysis—Blood
Whole blood samples, selected for analysis based on the amount of radioactivity present, were extracted with acetonitrile and analyzed by HPLC/RAM via an ion chromatographic separator. Conventional ion chromatography employs a fixed ion strength mobile phase with suppressed conductivity detection. However, this technique produced poor peak shapes for sulfate and fluorosulfate, which substantially lowered the detection limits. Therefore, the samples in this study were separated with an ion strength gradient and RAM detection. This gradient performed well with acceptable separation of two metabolites and good peak shape (Fig. 5).
Only two radiolabeled components were present in the blood extract samples. A 35S-sodium sulfate standard (Amersham Biosciences, Piscataway, NJ) was used to identify the first metabolite peak in the urine and blood profiles as 35S-sulfate by co-chromatography. The identification of the second peak as fluorosulfate was confirmed by 19F NMR spectroscopy.
Although not directly assayed, there was no evidence of parent 35S-sulfuryl fluoride in the blood based on radiochemical profiles. The presence of SO2F2 in blood was not expected, as work previously done in this laboratory showed, in vitro, rapid removal of SO2F2 from rat blood fortified with high levels of SO2F2 (t < 3 min) (unpublished data, The Dow Chemical Company).
A whole blood time-course of sulfate and fluorosulfate is presented in Figure 6. The amount of fluorosulfate is approximately 2-fold higher than sulfate at all sample times, except at 15 min after the beginning of the 300 ppm exposure, when the concentration of fluorosulfate is 6.5-fold higher, and 4 h after the end of the 300 ppm SO2F2 exposure, when only a small amount of fluorosulfate was detected. At all sample times, sulfate and fluorosulfate are approximately 3- to 5-fold higher after the 300 ppm exposure than after the 30 ppm exposure. Based on the limited amount of data available, a half-life for fluorosulfate elimination from whole blood was calculated to be 48 to 73 min, whereas the half-life for sulfate elimination from whole blood was calculated to be 50 to 64 min.
Chemical Analysis—Urine
Pooled urine samples, with selection and analysis of samples based on the amount of radioactivity present, were analyzed by HPLC/RAM. Two radioactive peaks tentatively identified as sulfate (by co-chromatography with 35S-sulfate) and fluorosulfate (by 19F NMR spectroscopy) were detected in urine (Fig. 5). The presence of SO2F2 in urine was not expected, as SO2F2 has been shown to be rapidly hydrolyzed in aqueous solutions (t < 18 min at pH = 8.07; Cady and Misra, 1974). During the exposure period, the amount of fluorosulfate eliminated in the urine was 3- to 3.5-fold higher than the amount of sulfate (Fig. 7). After the exposures, the amount of sulfate recovered in the urine was greater than the amount of fluorosulfate recovered in urine. Six to twelve h post-exposure, 5- to 7-fold more sulfate was recovered in the urine than fluorosulfate. The total μmoles of sulfate plus fluorosulfate recovered in the urine as determined by HPLC/RAM (3.8 and 43 μmoles; 30 and 300 ppm, respectively) compares well with the μmol-Eq SO2F2 in the urine + rinse as determined from the radiolabeled portion of this study (5.7 and 45 μmoles; 30 and 300 ppm, respectively).
Conversion of sulfate and fluorosulfate urine concentrations to rate estimates allowed calculation of half-lives for the elimination of sulfate and fluorosulfate in urine. Sulfate was eliminated in the urine with a half-life of 2.2 and 3.8 h for the 30 and 300 ppm exposure groups, respectively. Fluorosulfate was eliminated slightly faster, with a half-life estimate of 1.2 and 2.4 h for the 30 and 300 ppm exposure groups, respectively.
Fluoride Analysis
The ISE technique used in this study measures free fluoride only. Three types of fluoride make up the "total" fluoride level in tissues, ionic (free), acid labile, and nonionic (bound) (for a review, see Singer and Ophaug, 1982). Acid labile fluoride accounts for a very small amount of the total fluoride (0.01 ppm). However, the contribution of nonionic (bound) fluoride to the total fluoride level can be as high as 50% of the total in plasma. The determination of nonionic fluoride involves a very labor-intensive combustion method that provides highly variable results (ibid.), and therefore was not included in this study.
Ion selective electrode analysis of plasma and tissues was conducted with rats exposed to non-radiolabeled SO2F2 and with control rats exposed to clean air. Levels of plasma fluoride ion in control animals ranged from 0.023 to 0.070 μmol/ml plasma through a 24-h cycle. These measurements are within the control range of serum fluoride concentration reported by Eisenbrandt and Nitschke (1989).
An apparent slight elevation of plasma fluoride from control levels was observed during the 30 ppm and 300 ppm exposures, achieving concentrations of 0.046 and 0.132 μmol/ml plasma, respectively. But, plasma fluoride levels rapidly returned to control levels (0.03–0.05 μmol/ml plasma) by about 2 h after exposure was terminated (Fig. 6). Plasma fluoride was 1.6- and 5.4-fold higher than control levels at the end of exposure for the 30 and 300 ppm SO2F2 exposures, respectively. The maximum concentration of plasma fluoride measured at the termination of the 4-h 30 ppm SO2F2 exposure was 0.04 μmol/ml and is similar to that reported by Eisenbrandt and Nitschke (1989) following 6-h/day, 5 days/week for 13 weeks exposure to 30 ppm SO2F2, although considerable variation was reported. The plasma concentration reported here after the 300 ppm exposure was 0.13 μmol/ml and is nearly twice as large as that reported by Eisenbrandt and Nitschke (1989). However, our data indicate a rapid clearance of fluoride from the plasma and a return to background levels within 2 h following termination of exposure. Because serum was collected from the rats in the 13-week study at necropsy the day after the final exposure, measurement of serum fluoride was conducted subsequent to the time of peak levels of fluoride. Figure 6 presents the blood sulfate plus fluorosulfate data and the plasma fluoride data together in one figure for each exposure concentration. The overall shapes of the curves during and after the SO2F2 exposures are quite similar, indicating that the kinetics of formation and elimination from plasma of these three metabolites are similar or are interrelated.
Fluoride ion analysis of urine from non-exposed rats and from rats exposed to SO2F2 was conducted with selected urine samples. Figure 7 presents the urine sulfate plus fluorosulfate data and the fluoride data together in one figure for each exposure concentration. As observed with plasma, the overall shapes of the curves during and after the SO2F2 exposures are quite similar, indicating that the kinetics of formation and elimination of these three metabolites are similar or are interrelated.
Elevated levels of fluoride ion were detected in urine during and after the SO2F2 exposures. Non-exposed control rats had concentrations of fluoride ion of approximately 0.12–0.13 μmol/ml urine. By the end of the 4-h 30 ppm exposure, the urine levels of fluoride ion reached a maximum concentration of 0.5 μmol/ml urine. This concentration was maintained through 6 h post-exposure. By 12 h post-exposure the concentration had diminished to near background levels, 0.14 μmol/ml urine. In a similar fashion, by the end of the 4-h 300 ppm exposure the urine levels of fluoride ion reached a maximum concentration of 4.0 μmol/ml urine, about 8-fold higher than that obtained after the 30 ppm exposure. However, this concentration diminished through 6 h post-exposure to 1.7 μmol/ml urine and by 24 h post-exposure to 0.3 μmol/ml urine.
Fluoride levels in brain and kidney tissue during and after exposure to 30 and 300 ppm SO2F2 are presented in Figure 8. Kidney fluoride levels were roughly 2- to 2.5-fold higher at all collection times than those measured in control rats. Control rats had mean fluoride levels of 0.12 μmol/g kidney tissue, while both 30 and 300 ppm exposure levels resulted in mean fluoride concentrations of about 0.26 μmol/g kidney tissue. These levels were measured by the second hour of exposure and were maintained through 4 h post-exposure.
A slight 1.5-fold elevation in fluoride levels in brain tissue relative to control rats was observed during and after exposure to 30 ppm SO2F2. The mean control brain tissue fluoride ion concentrations were roughly 0.03 μmol/g tissue at all sacrifice times. Fluoride levels increased to 0.04 μmol/g brain after the 30 ppm SO2F2 exposure, whereas after the 300 ppm SO2F2 exposure, concentrations as high as 0.12 μmol/g tissue were measured, 4-fold higher than controls, at the end of the exposure period. The concentrations of fluoride in brain tissue approached the control levels by 4 h post-exposure.
DISCUSSION
Radiolabeled SO2F2 was rapidly absorbed via inhalation exposure, achieving maximum concentrations of 35SO2F2-derived radioactivity in both plasma and red blood cells near the end of the 4-h exposure periods. Saturation of absorption was not achieved during these 4-h exposures as evidenced by the following findings: (1) the measured levels of radioactivity were roughly proportional to the exposure concentrations, (2) the level of plasma/RBC radioactivity was increasing at the end of exposure, (3) steady-state 35S conditions were not achieved, and (4) the initial half-lives for clearance of radioactivity from plasma and RBC were the same at both exposure concentrations. Plasma levels of radioactivity during the 4-h exposure never reached steady-state conditions, most likely because of rapid elimination of radioactivity from the plasma. The similar peak concentrations of radioactivity measured with plasma and RBC suggests the 35S initially was evenly distributed between these two compartments. Once absorbed, the 35S was rapidly excreted, primarily via the urine. A large portion of absorbed 35S was excreted in the urine, even during the 4-h nose-only exposure.
Seven days post-exposure, small amounts of the radiolabel were evenly distributed among the tissues, suggesting incorporation into the tissues of 35S that had been transformed into amino acids; however, the chemical composition was not determined. Radioactivity was recovered mainly in respiratory tissues, which are the site of first exposure to the gas. The lungs had the highest levels of radioactivity 7 days post-exposure, and the nasal turbinates also had detectable radioactivity. Radioactivity associated with the RBC remained elevated 7 days post-exposure. Highly perfused tissues such as spleen and kidney also had higher levels of radioactivity than non-respiratory tissues, a finding attributed to the radioactivity in the blood. Radioactivity was rapidly cleared from plasma and RBC with initial half-lives of 2.5 h after the 30 ppm exposure and 1–2.5 h after the 300 ppm exposure. However, the terminal half-life of radioactivity was 2.5-fold longer in RBC than in plasma. The identification of fluorosulfate and sulfate in blood and urine suggests that SO2F2 is first hydrolyzed to fluorosulfate, with release of fluoride, followed by further hydrolysis to sulfate and release of the remaining fluoride. This apparent metabolism is supported by the increases in fluoride detected in the blood and urine following exposure of rats to SO2F2.
Fluoride metabolism resulting from inhalation exposure to SO2F2 would be expected to be consistent with the metabolism of fluoride from other sources. Whitford (1996) provides a comprehensive review of the metabolism of fluoride. Once absorbed, plasma functions as the "central compartment" for fluoride. Fluoride rapidly establishes a steady-state distribution between extracellular and intracellular fluids, and thus fluoride levels in these two compartments parallel each other.
The major route of fluoride elimination from the body is excretion in the urine. The kidneys are very efficient in removing fluoride from the body. The concentration of ionic fluoride in the glomerular filtrate is very similar to that of plasma, and there is no evidence for the tubular secretion of fluoride, although 62–78% of the filtered fluoride is reabsorbed. Approximately 50% of an absorbed amount of fluoride is excreted in the urine within 24 h, whereas most of the remainder becomes associated with calcified tissue.
Fluoride associated with calcified tissues is not irreversibly bound, and this is especially true of recently acquired fluoride on the surfaces of bone crystallites. If the intake of fluoride were to increase or decrease on a chronic basis, the concentrations in the calcified tissues would eventually reflect the change. Fluoride accumulation by bone is inversely related to age. The ability of the developing skeleton to clear fluoride from plasma more rapidly than the mature skeleton is largely a function of surface area (mature bone is compact, whereas the crystallites of developing bone provide extensive surface are for reactions involving fluoride). However, data in rats indicate that the uptake of fluoride by the skeleton continues throughout the first 18 months of life (75–85% of life span) and accounts for one half the plasma clearance during this period.
A likely cause of SO2F2 toxicity is the metabolic release of fluoride ions as postulated by Nitschke, et al. (1986) and Nitschke and Eisenbrandt (2001). The data presented here support the hypothesis that SO2F2 toxicity is the result of metabolic release of fluoride ions. Inhaled SO2F2 is rapidly absorbed and hydrolyzed to fluorosulfate and ionic fluoride, followed by further hydrolysis to sulfate and an additional fluoride ion. Therefore, these data suggest that the toxicity elicited by SO2F2 may be due to the release of fluoride ions, rather than a direct toxic action of SO2F2.
NOTES
The studies reported in this article were presented at the 42nd annual meeting of the Society of Toxicology, March 2003, Salt Lake City, UT (The Toxicologist 72, 713, March 2003).
VIKANE is a trademark of the Dow AcroSciences LLC.
ACKNOWLEDGMENTS
The authors thank T. Card, A. J. Clark, J. Y. Domoradzki, M. J. Filary, J. A. Hotchkiss, C. E. Houtman, M. A. Knoerr, S. M. Krieger, G. T. Marty, L. G. McFadden, E. S. Mullen, D. L. Rick, S. Saghir, and C. M. Thornton for their excellent technical assistance. Conflict of interest: The Dow Chemical Company, Dow AgroSciences LLC.
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