Introduction

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CFCs¹ are used as refrigerants, propellants, and degreasing and dry-cleaning agents. Because of their chemical stability, CFCs may migrate to the stratosphere and cause stratospheric ozone depletion (Molina and Rowland, 1974), which may increase ultraviolet radiation on the earth's surface and cause adverse human health effects (Longstreth, 1988; Taylor et al., 1988). These concerns have prompted international limits on production and use of CFCs.

HCFCs are being developed as replacements for ozone-depleting CFCs. The presence of C-H bonds in the HCFC molecules makes them susceptible to oxidation in the troposphere and thereby reduces HCFC migration to the stratosphere (Anders, 1991; Dekant, 1996). Hence, HCFCs have less ozone-depleting potential than CFCs.

HCFC-141b is being developed as a CFC replacement. The metabolic fate and gas-uptake pharmacokinetics of HCFC-141b have been studied in rats. HCFC-141b is metabolized to 2,2-dichloro-2-fluoroethanol, which is conjugated with glucuronic acid and excreted in the urine (Harris and Anders, 1991). Gas-uptake pharmacokinetic studies show that HCFC-141b uptake by rats is a first-order process and that 2,2-dichloro-2-fluoroethyl glucuronide excretion is exposure concentration-dependent (Loizou and Anders, 1993; Loizou et al., 1996). The no-observable-effect level of HCFC-141b in acute and subchronic toxicity studies is 8,000 ppm (Brock et al., 1995). In inhalation teratology and two-generation reproduction studies, the no-observable-effect level is 2,000 ppm for all indices (Rusch et al., 1995). HCFC-141b is not genotoxic in a battery of in vitro tests, and the no-observable-effect level for chronic toxicity is 1,500 ppm (Millischer et al., 1995).

The use of HCFC-141b may be accompanied by human exposure. The present studies were undertaken, therefore, to study the metabolism of HCFC-141b in human subjects exposed by inhalation. The results show that HCFC-141b undergoes exposure concentration-dependent metabolism to 2,2-dichloro-2-fluoroethanol, which is excreted as its glucuronide.

	Materials and Methods

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Materials. HCFC-141b (99.88% pure, lots ES5076B and ES3148A) was provided by AlliedSignal Inc. (Morristown, NJ). Lithium aluminum hydride, methyl trichloroacetate, antimony (III) fluoride, tetrahydrofuran, and diethyl ether were obtained from Aldrich Chemical Co. (Milwaukee, WI). -Glucuronidase, type B-3, was purchased from Sigma Chemical Co. (St. Louis, MO). 2,2-Dichloro-2-fluoroethanol was obtained by synthesis (see below).

Syntheses. 2,2-Dichloro-2-fluoroethanol was synthesized by reduction of methyl dichlorofluoroacetate with lithium aluminum hydride.

Methyl dichlorofluoroacetate. Methyl dichlorofluoroacetate was synthesized as previously described by Ishihara and Kuroboshi (Ishihara and Kuroboshi, 1987). Briefly, methyl trichloroacetate (26.6 g, 0.15 mol) and antimony (III) fluoride (17.9 g, 0.1 mol) were mixed in a flask equipped with a distillation head and heated to about 130^oC in oil bath. Bromine (5.2 ml, 0.1 mol) was slowly added to the stirred mixture, which was heated and stirred for 30 min. Distillation of the product gave methyl dichlorofluoroacetate that was 98% pure by ¹H NMR and ¹⁹F NMR spectral analysis. The major impurity was methyl chlorodifluoroacetate.

2,2-Dichloro-2-fluoroethanol. Lithium aluminum hydride (3.1 ml of a 1 M tetrahydrofuran solution, 0.5 equiv., equal to 2.0 equiv. of hydride ion) was added slowly with stirring over 30 min to methyl dichlorofluoroacetate (1g, 6.2 mmol) in 20 ml of dry tetrahydrofuran at 15^oC. After the addition was complete, the mixture was stirred at 15^oC for 2 hr. Excess lithium aluminum hydride was decomposed by addition of 115 µl water, followed by 115 µl of 15% sodium hydroxide and then another 345 µl water. The mixture was stirred for about 15 min and then filtered under vacuum. The filtrate was concentrated under reduced pressure to remove tetrahydrofuran. Distillation gave 2,2-dichloro-2-fluoroethanol, which was 92% pure by ¹H NMR and ¹⁹F NMR spectrometry. The mass spectrum was identical with that reported previously (Harris and Anders, 1991). The major impurities were tetrahydrofuran and 2-chloro-2-fluoroethanol.

Instrumental Analyses. NMR spectra were acquired with a Bruker WP270 spectrometer Bruker Instruments, Inc., Billerica, MA) operating at 270.13 MHz for ¹H and at 254 MHz for ¹⁹F. Chemical shifts were referenced to tetramethylsilane in CDCl₃ ( = 0) for ¹H NMR spectra or to trifluoroacetamide in CDCl₃ or D₂O ( = 0 ppm) for ¹⁹F spectra. GC/MS analyses were performed with a Hewlett-Packard 5890 gas chromatograph (Hewlett-Packard, Palo Alto, CA) equipped with a DB-Wax column (30 m × 0.25 mm i.d., 0.5-µm film thickness; J & W Scientific, Folson, CA) and coupled to a HP-5970 mass selective detector. The temperatures of the injection port and detector were 125^oC and 230^oC, respectively. Samples were analyzed with a temperature program of 100^oC for 1 min, followed by a linear gradient of 10^oC/min to 220^oC and then a constant temperature at 220^oC for 3 min.

Subjects and Exposures. Volunteers who had no cardiopulmonary disease, no history of smoking, no recent upper respiratory illness, no abnormal pulmonary or liver function, as determined by history and physical examination, were selected. The subjects ranged in age from 22-30 years and included both males and females. All female subjects had negative pregnancy tests. The study was reviewed and approved by the Research Subjects Review Board of the University of Rochester, and informed consent was obtained from all subjects.

Analysis of Urinary 2,2-Dichloro-2-fluoroethyl Glucuronide. GC/MS. The method previously described was modified to quantify urinary 2,2-dichloro-2 fluoroethanol concentrations (Harris and Anders, 1991). Urine samples (10 ml) were lyophilized and reconstituted in 2 ml of 0.1 M acetate buffer (pH 5.0) and incubated overnight at 37°C with -glucuronidase (10 mg). The internal standard 2,2,2-trichloroethanol (15.57 µg) was added to the hydrolyzed samples, which were extracted with 5 ml of diethyl ether. The phases were separated by centrifugation, and the ether layer was transferred to a clean tube and then concentrated under a stream of dry nitrogen at room temperature for GC/MS analysis. The areas corresponding to 2,2-dichloro-2-fluoroethanol and 2,2,2-trichloroethanol were measured by selective-ion monitoring, and the ratio of the areas was used to quantify the concentration of 2,2-dichloro-2-fluoroethanol. Fragment ions at m/z 77 and 97 were measured for 2,2-dichloro-2-fluoroethanol and at m/z 77, 82, and 113 for 2,2,2-trichloroethanol. The retention times for 2,2-dichloro-2-fluoroethanol and 2,2,2-trichloroethanol were 5.5 and 7.6 min, respectively. 2,2-Dichloro-2-fluoroethanol concentrations were measured with a standard curve prepared with the synthetic standard under the same conditions, which was normalized against its purity. The detection limit was 0.2 nmol/ml urine.

¹⁹F NMR spectroscopy. A representative urine sample (25 ml) from each subject exposed to HCFC-141b was lyophilized and dissolved in 2 ml of 0.1 M acetate buffer (pH 5.0) and centrifuged. The supernatant was transferred to two NMR tubes (0.8 ml/tube). Deuterium oxide (100 µl) was added to one tube for ¹⁹F NMR analysis. -Glucuronidase (10 mg) dissolved in 100 µl of D₂O was added to the other tube, and the mixture was incubated overnight at 37°C. The unhydrolyzed and hydrolyzed reaction mixtures were analyzed by ¹⁹F NMR spectroscopy.

	Results and Discussion

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Analysis by ¹⁹F NMR spectroscopy of urine of human subjects exposed to 1,000 ppm HCFC-141b showed resonances that were assigned to 2,2-dichloro-2-fluoroethyl glucuronide (fig. 1A). Two minor metabolites were also observed in unhydrolyzed human urine samples (fig. 1A). One of the resonances ( = 17.5 ppm, singlet) was assigned to dichlorofluoroacetic acid, which is a known metabolite of HCFC-141b in rats (Harris and Anders, 1991). The other resonance was not assigned. After incubation of the urine samples with -glucuronidase, the resonances assigned to 2,2-dichloro-2-fluoroethyl glucuronide were lost and new resonances that were assigned to 2,2-dichloro-2-fluoroethanol appeared (fig. 1B). The ¹⁹F NMR spectrum of synthetic 2,2-dichloro-2-fluoroethanol dissolved in concentrated human urine from unexposed subjects was not changed after incubation with -glucuronidase, indicating that 2,2-dichloro-2-fluoroethanol is stable under the analytical conditions employed. These data show that HCFC-141b is metabolized to 2,2-dichloro-2-fluoroethanol, which is conjugated with glucuronic acid and excreted in the urine. Previous studies show that 2,2-dichloro-2-fluoroethanol is the major metabolite of HCFC-141b and that dichlorofluoroacetic acid is a minor metabolite in the rat (Harris and Anders, 1991; Loizou and Anders, 1993). Thus the metabolic fate of HCFC-141b in human subjects is qualitatively similar to the pathway established in the rat.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   19F NMR spectra of a representative urine sample of a human subject exposed to 1,000 ppm HCFC-141b. Panel A, before hydrolysis with -glucuronidase; Panel B, after hydrolysis with -glucuronidase.

2,2-Dichloro-2-fluoroethyl glucuronide was detected in all subjects exposed to HCFC-141b. Unconjugated 2,2-dichloro-2-fluoroethanol was not detected in urine samples before enzymatic hydrolysis, indicating that 2,2-dichloro-2-fluorethanol was excreted solely as its glucuronide, as shown by the NMR spectral data (fig. 1). In addition, 2,2-dichloro-2-fluoroethanol was not detected in the hydrolyzed urine samples collected before exposure.

Exposure concentration- and time-dependent excretion of 2,2-dichloro-2-fluoroethyl glucuronide was observed. 2,2-Dichloro-2-fluoroethyl glucuronide concentrations were highest in the samples collected from 4 to 12 hr after exposure, but the conjugate was also detected in samples collected from 0 to 4 hr or 12 to 24 hr after exposure (table 1). Concentration-dependent excretion of 2,2-dichloro-2-fluoroethyl glucuronide was observed in all exposed human subjects, except EB (table 1), but data are not available that allow estimation of the fraction of the retained HCFC-141b that was metabolized to 2,2-dichloro-2-ethanol. Exposure concentration-dependent excretion of 2,2-dichloro-2-fluoroethyl glucuronide is also seen in rats exposed to 5,000 to 20,000 ppm HCFC-141b for 6 hr (Loizou and Anders, 1993).

In summary, 2,2-dichloro-2-fluoroethanol, which was excreted as its glucuronide in urine, was the major metabolite of HCFC-141b in humans. Excretion of 2,2-dichloro-2-fluoroethyl glucuronide was dose-dependent in humans exposed to 250, 500, and 1,000 ppm HCFC-141b, and dichlorofluoroacetic acid was a minor metabolite.