Metabolism of the hydrochlorofluorocarbon 1, 2-dichloro-1, 1

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Chem. Res. Toxicol. 1991, 4 , 180-186

180

Metabolism of the Hydrochlorofluorocarbon 1,2-Dichloro-I ,l-difluoroethane James W. Harris and M. W. Andem* Environmental Health Sciences Center and Department of Pharmacology, University of Rochester School of Medicine, 601 Elmwood Avenue, Rochester, New York 14642 Received October 23, 1990

1,2-Dichloro-l,l-difluoroethane (HCFC-132b) is a potential substitute for some ozone-depleting chlorofluorocarbons and a model for other 1,1,1,2-tetrahaloethanesunder consideration as chlorofluorocarbon substitutes. Male Fischer 344 rats were given 10 mmol/kg HCFC-132b dissolved in corn oil by intraperitoneal injection. An NMR assay for covalent binding of HCFC-132b metabolites to liver proteins was negative, whereas binding was observed in halothane-treated rats. Total urinary metabolites excreted by rats given HCFC-132b during the first 24 h amounted to 1.8 f 0.1% of the injected dose, as determined by 19FNMR. During glucuronide, the first 6 h, metabolites of HCFC-132b corresponding to 2-chloro-2,2-difluoroethyl unknown metabolite A, chlorodifluoroacetic acid, and chlorodifluoroacetaldehyde hydrate [both free and conjugated (unknown metabolite B)] were excreted in urine in the approximate ratio 100.93:7, respectively. Metabolite A is apparently an O-conjugate of 2-chloro-2,2-difluoroethanol; unconjugated 2-chloro-2,2-difluoroethanol was not detected in urine. The 19FNMR spectrum of metabolite B indicates the formation of a hemiacetal of chlorodifluoroacetaldehyde. Repeated exposure of rats to HCFC-132b significantly increased both the rate of chlorodifluoroacetic acid excretion and the relative fraction of the HCFC-132b dose excreted as chlorodifluoroacetic acid in urine. Incubation of HCFC-132b with rat hepatic microsomes yielded chlorodifluoroacetaldehyde hydrate as the only fluorinated product. The in vitro metabolism of HCFC-132b was increased in microsomes from pyridine-treated rats as compared with control rats, and HCFC-132b metabolism was inhibited by p-nitrophenol, indicating that the cytochrome P-450 isoform I I E l is largely responsible for the initial hydroxylation of HCFC-132b.

Introduction Hydrochlorofluorocarbons (HCFCs)' are being developed as substitutes for chlorofluorocarbons (CFCs) that deplete stratospheric ozone ( I ) . Restrictions on the production of CFCs mandated by the Montreal Protocol have stimulated an urgent search for CFC substitutes (2). HCFCs, which possess many of the useful physical properties of CFCs, degrade in the troposphere before reaching the stratosphere and show little ozone-depletingpotential (3). However, the tropospheric lability imparted by the presence of C-H bonds in HCFCs also makes them excellent candidates for oxidative metabolism, in contrast to CFCs ( 4 , 5 ) . Although HCFCs will be introduced into commerce soon, little toxicity data are available for these compounds (5). Because HCFCs will be used widely, there may be significant global human exposure to HCFCs. Therefore, it is important that the metabolism and toxicities of HCFCs be fully evaluated. Most CFC substitutes are l,l,l-trihaloethanes (CX3CH3, X = halogen), 1,1,1,2-tetrahaloethanes(CX3CH2X),or pentahaloethanes (CX3CHX,). 1,2-Dichloro-l,l-difluoroethane (HCFC-l32b), l,l,l-trifluoro-2-chloroethane (HCFC-l33a),and 1,1,1,2-tetrafluoroethane(HCFC-134a) are being considered as substitutes for dichlorodifluoromethane (CFC-12) (6, 7),whose 1989 US. production amounted to 1.8 X lo8 kg. HCFC-132b undergoes dechlorination when incubated with rat hepatic microsomes (81, but no organic metabolites have been identified. The metabolism of the fluorinated analogue HCFC-134a has been studied: HCFC-134a undergoes defluorination when incubated with rat hepato-

* Correspondence should be addressed to this author.

cytes or hepatic microsomal fractions (9, IO). A preliminary toxicity study of HCFC-132b given to rats by inhalation revealed proliferation of bile duct epithelial cells, testicular damage, elevated liver, heart, kidney, and lung to body weight ratios, decreased brain and testis weights, and low activity and responsiveness to noise (11). We have chosen HCFC-132b as a model compound for the study of 1,1,1,2-tetrahaloethanemetabolism and report herein the results of studies on its in vivo and in vitro metabolic fate.

Experimental Procedures Spectroscopy. Fluorine NMR spectra were obtained with a

Bruker WP-270 instrument equipped with a 5-mm dedicated 'q probe and operating at 254.18 MHz for fluorine. The pulse width was 3 ps, and the interpulse time was 1.7 s for metabolite quantification (spectral width = 5 kHz) or 0.7 s for covalent binding assays (spectral width = 50 kHz). Spectra were acquired a t room temperature with sample spinning. For the assay of covalent binding, at least 35 000 transients were acquired. Chemical shifts and signal integrals were referenced to a 5.0 mM solution of trifluoroacetamide in D20(6 = 0 ppm) placed in a sealed coaxial tube. The method of Hull (12) was used for quantification of metabolites by 'gF NMR. Briefly, this method requires that the entire spectral width of interest be uniformly excited by the radio-frequency pulse. This may be assessed with a single reference signal by acquiring a fixed number of transients for each of n transmitter offset values that span the spectral width of interest. When all transients are accumulated into one free induction decay, the resulting composite spectrum should contain n reference signals with equivalent line shapes and integrals.

' Abbreviations: CFC, chlorofluorocarbon; HCFC, hydrochlorofluorocarbon; HCFC-l32b, 1,2-dichloro-1,l-difluoroethane; HCFC-l34a, l,l,l,Z-tetrafluoroethane; PNP, p-nitrophenol. 0 1991 American Chemical Society

Metabolism of HCFC-132b Further, sufficient relaxation times are necessary t o correct for between the differences in spin-lattice relaxation times (TI) various metabolites. The lBFNMR spectra of urinary and microsomal metabolites were recorded in H2O/D,O solution. Mass spectra were recorded with a Hewlett-Packard 5880A GC equipped with an HP-1 (dimethylpolysiloxane gum) capillary column and coupled to an HP-5970 mass-selective detector (70 eV, electron impact). Chemicals, HCFC-132b was purified from the commercial product (PCR Inc., Gainesville, FL; 4% HCFC-122 and 1% HCFC-142b were present as impurities) by distillation through a 20-cm Vigreaux column. Several fractions boiling a t 46-47 "C were collected, but only the middle fractions, which were pure by GC-MS and by 19F NMR analysis, were used. Chlorodifluoroacetic acid and methyl chlorodifluoroacetate were obtained from PCR Inc. and were pure as assessed by 19FNMR. All other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI). Enzymes and cofactors were obtained from Sigma Chemical Co. (St. Louis, MO). Chlorodifluoroacetaldehyde ethyl hemiacetal [MS: m / z (re1 intensity) 145 (1.6), 133 (1.2), 131 (2.2), 117 (1.4), 115 (4), 87 (7.9), 85 (28), 75 (loo), 67 (25); 'gF NMR (CDClJ: AB spectral pattern, 4.77 and 3.93 ppm, doublet of doublets, 2JFF = 167 Hz, 3JHF = 4.1 Hz] was synthesized according to the method of Yamada et al. (13). 2-Chloro-2,2-difluoroethanol [MS: m / z (re1 intensity) 118 (0.3), 116 (0.7), 87 (5.0), 85 (16), 81 (loo), 61 (81), 51 (34)] was obtained as a side product of this reaction. A mixture of chlorodifluoroacetaldehyde ethyl hemiacetal and 2-chloro-2,2difluoroethanol, which were not separable by simple distillation, was used as a reference standard for metabolite identification. Chlorcdifluoroacetaldehyde [MS: m/z (re1intensity) 116 (8.4), 114 (20), 87 (8.4), 85 (20), 79 (86), 69 (23), 68 (9.2), 67 (33), 66 (15), 51 (loo)] was obtained by hydrolysis of the ethyl hemiacetal in water (13). In Vivo Experiments. (A) Animal Treatment. Male Fischer 344 rats (Charles River, 230-300 g) were given 10 mmol/kg HCFC-132b dissolved in corn oil by intraperitoneal (ip) injection. A short period of anesthesia was observed with most animals. The treated animals were immediately placed in metabolism cages and kept on a 12-h light/dark cycle. Urine was collected into vessels maintained at -78 "C and was stored frozen until analyzed. Some rats were given three additional 10 mmol/kg doses of HCFC-132b ip, as indicated in Figure 6A. (B) Assay for Covalent Binding of HCFC-132b Metabolites. Fifteen hours after receiving a single dose of HCFC-l32b, rats were anesthetized with ether, and the livers were excised. Microsomal and cytosolic fractions were prepared by differential centrifugetion: the supernatant from a 10000g, 20 min, centrifugation of the liver homogenate was centrifuged for 60 min a t looOOOg, yielding the cytosolic fraction as the supernatant and the microsomal fraction as the pellet. The fractions were suspended in water and placed in dialysis tubing (Spectrapor 3,3500 MW cutoff). Equilibrium dialysis in 10 mM phosphate buffer, pH 7.0, containing 0.1% (w/v) sodium dodecyl sulfate (SDS) was employed to remove weakly bound metabolites (14). The dialysis was carried out with constant stirring a t 4 "C; the buffer was changed several times during at least 48 h of SDS dialysis. After dialysis, the protein was lyophilized and then dissolved in D 2 0 for '9 NMR analysis. Protein concentrations in the NMR tube were kept as high as pmaible while still maintaining a liquid, albeit viscous, solution. A minimum of 25 mg of protein/mL of D 2 0 was used. Protein concentrations were measured with a Bio-Rad (Richmond, CA) protein assay kit with bovine serum albumin as the standard. Livers from rats given 10 mmol/kg halothane ip were similarly treated. In Vitro Experiments. (A) Hepatic Microsomes. Male Fischer 344 rats (260-320 g) were used. In some experiments rats were given 100 mg/kg pyridine dissolved in 20 mM phosphate buffer, pH 7.0, ip once daily for 4 days. This treatment increases hepatic concentrations of cytochrome P-450 isoform I I E l (15). Induced rats were fasted for 12 h after the final pyridine treatment, and their livers were removed while under ether anesthesia. Livers were homogenized in a Dounce apparatus a t 4 "C in 20 mM phosphate buffer, pH 7.4, containing 1.0 M KCl and 10 mM ethylenediaminetetraacetic acid (buffer A), and the homogenate was centrifuged at loooOg for 20 min. The resulting supernatant

Chem. Res. Toxicol., Vol. 4, No. 2, 1991 181 was centrifuged at lOOOOOg for 60 min. The microsomal pellet was resuspended in buffer A and centrifuged again a t lOOOOOg for 60 min. The resulting microsomes were suspended in 0.10 M phosphate buffer, pH 6.8, a t a protein concentration of 4.0 mg/mL. Protein concentrations were measured as described above. Microsomes from untreated animals were similarly prepared. (B) Microsomal Incubations. Incubation mixtures contained freshly isolated microsomes, substrates or inhibitors t19 noted, a NADPH-generating system (final concentrations: 9 mM glucose 6-phosphate, 1 mM NADP+, 0.2 unit/mL glucose-6-phosphate dehydrogenase), and 0.1 M phosphate buffer, pH 6.8. All incubations were conducted at 37 "C in a shaking water bath. At least two replicates of the following incubations were conducted with each batch of microsomes. When HCFC-132b was the substrate, the incubation mixtures were contained in reaction flasks sealed with Teflon-lined septa. Final concentrations of HCFC-132b and microsomes were 5.0 mM and 2.0 mg/mL, respectively. The time course of the reaction was assessed by withdrawing 0.50-mL samples from the flask with a syringe, adding the sample to an ice-cold, 2-mL reaction vial, sealing the vial with a Teflon cap, and heating the vial a t 100 "C for 5 min. Product formation was assayed by quantitative 19F NMR. Hydroxylation of p-nitrophenol (PNP) was measured as a selective assay for cytochrome P-450 IIEl activity (1516). Final concentrations of P N P and microsomes were 450 r M and 1.0 mg/mL, respectively. The time course of the reaction was assessed by adding 0.5-mL samples from the reaction mixture to 0.25 mL of 0.6 N perchloric acid in a 1.5-mL plastic centrifuge tube. After centrifugation to precipitate proteins, a sample (0.70 mL) of the resulting supernatant was made alkaline by adding 70 pL of 10 M NaOH, and the absorbance at 546 nm was recorded. Care must be taken to measure product formation immediately after base addition.2 Incubation mixtures containing both P N P and HCFC-132b were enclosed in sealed reaction flasks. Final concentrations of PNP, HCFC-l32b, and microsomes were 300 pM,5.0 mM, and 2.0 mg/mL, respectively. Reaction mixtures were incubated at 37 "C for 20 min. At 20 min, the enzymatic reaction was stopped by adding half of the mixture t o one-half volume of 0.6 N perchloric acid for subsequent assay of P N P hydroxylation and by heating the other half of the reaction mixture in a closed vial a t 100 "C for 5 min for subsequent 'V NMR analysis of HCFC-132b metabolites. Identification of Urinary Metabolites. Examination of urine from HCFC-132b-treated rats revealed the presence of several fluorine-containing metabolites. Hence experiments were conducted t o identify these metabolites. The formation of 2-chloro-2,2-difluoroethyl glucuronide as a metabolite of HCFC-132b was investigated by incubating 0.25 mL of urine from HCFC-132b-treated rats, 0.25 mL of D20, and 5 mg of @-glucuronidase(type V-A) in an NMR tube at room temperature. Spectra were taken both before addition of 8-glucuronidase and a t various times afterward. In some experiments, saccharic acid l,l-lactone, a competitive inhibitor of @-glue uronidase (13,was added (10 mM final concentration) 3 h after the addition of &glucuronidase. Attempts were made to cleave metabolite A by incubating urine of HCFC-132b-treated rats with sulfatase (Sigma type H-1; EC 3.1.6.1), esterase (Sigma type 1; EC 3.1.1.1), alkaline phosphatase (Sigma type XXXII; EC 3.1.3.1), and @-glucosidase(Sigma type 1; EC 3.2.1.20). The presence of enzyme activity was confirmed in each of these incubations. Headspace GC-MS analysis of urine from HCFC-132b-treated rats was used to identify chlorodifluoroacetic acid as a metabolite (18). The methyl chlorodifluoroacetate formed by sulfuric The hydroxylation of PNP to form 4-nitrocatecholhas been used as a selective assay for microsomal cytochrome P-450 IIEl (15,16). However, with the standard assay protocol (16), a rapid loss of measured absorbance at 546 nm was observed after base addition. Authentic 4nitrocatachol, the presumed product of IIEl hydroxylation of PNP (16), showed no loss of absorbance under identical conditions. Studies on the validation of PNP hydroxylation for the assay of cytochrome P-450 IIEl activity are in progress.

Harris and Anders

182 Chem. Res. Toxicol., Vol. 4, No. 2, 1991

A .. . .

I

.. ..

20

-...-.

, .

.,.

10

0

-10

-20

PPH

Figure 1. 19FNMR spectra of dialyzed liver microsomal proteins from rats given 10 mmol/kg HCFC-132b (A) or halothane (B). The broad resonance in panel B has been identified as protein-bound Nf-(trifluoroacety1)lysine (19). Livers from untreated rats showed no resonances, as in panel A.

I

I

-

2Chloro-2~difluorwlhyl glucuronide

Melabolite A

Chlorcdifluomacclaldehyde Conjugated (Metabolite B)

J

Chlorodifluoroacelic acid

acid-dimethyl sulfate treatment of urine (18) showed a mass spectrum identical with that of an authentic sample. Furthermore, the singlet in the lgF NMR spectrum of authentic chlorodifluoroacetic acid coresonated with that found in urine. Chlorodifluoroacetaldehydehydrate was identified in urine by its 19FNMR chemical shift, spectral pattern, and coupling constant (Table I), which were identical with those of an authentic sample. Synthetic chlorodifluoroacetaldehyde ethyl hemiacetal (see above) and conjugated chlorodifluoroacetaldehydehydrate (metaboliteB, Table I) displayed similar AB 19FNMR spectral patterns.

Results In Vivo Experiments. No covalent binding of fluorinated metabolites of HCFC-132b to liver proteins was detected by 19F NMR (Figure l A , n = 3). A covalent adduct was detected, however, in livers of rats given an equimolar dose of halothane, whose metabolism is known to produce trifluoroacetyl chloride ( 4 ) (Figure 1B). This halothane adduct has been identified as N'-(trifluoroacety1)lysine (I9). Figure 2 shows the NMR spectrum of all urinary metabolites of HCFC-132b detected in this study. Spectra were acquired over a span of 350 ppm, which encompasses the resonances of organofluorine compounds (20),thus ensuring that these are the only fluorinated metabolites excreted in detectable quantities. The 19FNMR spectral values for each metabolite are shown in Table I. The resonances centered at 13.30 ppm were assigned to 2-chloro-2,2-difluoroethyl glucuronide. Incubation of urine with @-glucuronidaseresulted in the loss of the resonances at 13.30 ppm and the appearance of a triplet at 11.58 ppm (Figure 3) that was identical with a synthetic standard of 2-chloro-2,2-difluoroethanol.No cleavage of the gluc-

Hydrdte

\

Table I. 19F NMR Data for HCFC-132b Metabolites' ~___________ multicoupling metabolite 6, ppm plicit? constants 2-chloro-2,2-difluoroethyl 13.30 and t 3JHp 11.3 HZ 13.32 glucuronide metabolite A 13.05 t 3JHF = 11.3 HZ 12.71 9 chlorodifluoroacetic acid 2-chloro-2,2-difluoro11.58 t 3JHF E 11.3 HZ ethanol metabolite B AB system; d of d ~ J F=F167 Hz; 8.48 and 3 J ~ = p 6 HZ ~~

7.66

chlorodifluoroacetaldehyde hydrate

5.56

d

3JHp

= 4.3 HZ

Chemical shifts are reported aa ppm downfield from trifluoroacetamide (6 = 0). leF NMR spectra were recorded as described under Experimental Procedures. b s = singlet, d = doublet, t = triplet.

uronide occurred in the absence of enzyme. The enzymatic cleavage of the glucuronide was inhibited by 10 mM saccharic acid 1,Clactone (Figure 3C), which is a competitive inhibitor of &glucuronidase (17). The complexity of the 19F NMR spectrum of 2-chloro-2,2-difluoroethyl glucuronide, which is characterized by two 3 J = ~11.3 ~ Hz triplets separated by 4 Hz (Figures 2 and 3), can be explained by second-order spectral interactions (21). The presence of chlorodifluoroacetic acid in urine of HCFC-132b-treated rats was confirmed by headspace GC-MS analysis of the methyl ester formed by acidic dimethyl sulfate treatment of urine (18) and by the demonstration of NMR coresonance of the metabolite with authentic chlorodifluoroacetic acid (data not shown). The 19F NMR spectrum of metabolite A (Figure 2) shows a triplet with J H F = 11.3 Hz, which is consistent with vicinal coupling to two protons. This spectral pattern

Chem. Res. Toxicol., Vol. 4, No. 2, 1991 183

Metabolism of HCFC-132b

t

I Glucuronide " "

'

.

"

" " '

J

LF l'

Chlorodifluoroacetic acid

13.0

PPM

12.0

Figure 3. 8-Glucuronidase treatment of urine from HCFC132btreated rats. (A) Urine before enzyme addition; (B) 3 h after enzyme addition and immediately after adding saccharic acid 1,l-lactone;(C)spectrum B recorded 3 days after enzyme addition. In the absence of saccharic acid 1,4-lactone,conversion of the (11.6 ppm) was glucuronide to free 2-chloro-2.2-difluoroethanol complete within 24 h.

Chlorodifluoroacetaldehyde: Hydrated and conjugated (Metabolite 6 )

0

5

!mol

10

15

20

25

30

Metabolite Excreted

Figure 5. Urinary excretion of 2-chloro-2,2-difluoroethylglucuronide, metabolite A, chlorodifluoroacetic acid, chlorodifluoroacetaldehyde hydrate, and metabolite B after giving a single 10 mmol/kg dose of HCFC-132b. (Hatched bars) 0-6 h, (stippled bars) 6-12 h, (solid bars) 12-24 h. Data are shown as means f SD (n = 3).

Figure 4. Expanded '@FNMR spectrum of urine from an HCFC-132b-treatedrat showing the characteristic AB spectral pattern of chlorodifluoroacetaldehydehemiacetal (metaboliteB). indicates that metabolite A may be an 0-conjugate of 2-chloro-2,2-difluoroethanol. 2-Chloro-2,2-difluoroethanol was released from metabolite A by treatment with 10% concentrated HC1 and by heating a t 100 "C for 1.5 h. Metabolite A was not cleaved by @-glucosidase,sulfatase, alkaline phosphatase, or esterase. Metabolite A could not be extracted from urine with ethyl ether, indicating that it is relatively polar. Unconjugated 2-chloro-2,2-difluoroethanol was not detected in the urine of HCFC-132b-exposed rats. Chlorodifluoroacetaldehyde (Figure 2) was present in urine in two forms: an unconjugated hydrate and an apparently conjugated form (metabolite B), which can be hydrolyzed to the hydrate. The 19F NMR spectrum of metabolite B displays an AB spectral pattern that is more complex (Figure 4) than the doublet of chlorodifluoroacetaldehyde hydrate, indicating that one of the hydrate hydroxyl groups on C-1 is conjugated, thus making C-1 chiral, and that the two C-2 fluorines are, therefore, magnetically nonequivalent. The spectral patterns of synthetic chlorodifluoroacetaldehydeethyl hemiacetal and metabolite B were similar, and both were hydrolyzed to give chlorodifluoroacetaldehyde hydrate. Further, mass

spectral analysis of metabolite B gave fragment ions similar to those of synthetic chlorodifluoroacetaldehyde ethyl hemiacetal, but a molecular ion was not obtained. Total urinary metabolites excreted during the first 24 h after giving a single 10 mmol/kg dose of HCFC-132b to rats amounted to 1.8 f 0.1% (n = 3) of the injected dose. The amounts of the several HCFC-132b metabolites excreted during this same time period are presented in Figure 5. During the first 6 h, for example, metabolites of HCFC-132b corresponding to 2-chloro-2,2-difluoroethyl glucuronide, metabolite A, chlorodifluoroacetic acid, and chlorodifluoroacetaldehyde hydrate (free and conjugated) were excreted in urine in the approximate ratio of 100.937, respectively. Urinary metabolite excretion was also measured in rats given repeated 10 mmol/kg doses of HCFC132b. By 24 h, the excretion of chlorodifluoroacetic acid as a proportion of total metabolites increased (Figure 5). With repeated exposure to HCFC-l32b, the fraction of total metabolites represented by chlorodifluoroacetic acid increased markedly (Figure 6A), and the rate of urinary chlorodifluoroacetic acid excretion also increased (Figure 6B). In Vitro Experiments. Chlorodifluoroacetaldehyde hydrate was the only fluorinated metabolite detected when HCFC-132b was incubated with microsomes from control or pyridine-induced rats. Product formation was linear with time for 20 min with an apparent rate of 4.3 nmol/ (min-mg of protein) with microsomes from pyridine-induced rats (data not shown). Microsomes from control rats produced detectable amounts of the aldehyde hydrate (approximately 10 nmol) only after 20 min of incubation (Figure 7). PNP metabolism was increased 4.8-fold in microsomes from pyridine-treated rats versus controls (Figure 7) and was time-linear for 20 min with both

184 Chem. Res. Toxicol., Vol. 4, No. 2, 1991

Harris and Anders

A 80

T 100-

60 I+

80 -

a

JJ

0

40

60 -

cu 0

*

40 -

20

20 -

0 50

0

100

150

n-

Time ( h )

B

1.2 Figure 7. Comparative rates of PNP and HCFC-132b hydroxylation by hepatic microsomes prepared from pyridine-treated (n = 3) (stippled bars) and control (n = 2) (solid bars) rats (20-min incubations). Rates of PNP metabolism were calculated with the molar absorptivity reported for 4-nitrocatechol Data are shown as means f SD.

1.0

0.8

0.6 0.4

0.2 nn

u*u

0

-6

6

- 12

12

- 24

24

- 51

51

- 80

Time (h) Figure 6. Urinary excretion of chlorodifluoroacetic acid after repeated treatment with HCFC-l32b, expressed as a fraction of total metabolites excreted (A) or as the rate of excretion (B). Arrows in panel A indicate times when a 10 mmol/kg dose of HCFC-132b was given ip. Data are shown as means SD (n = 3).

*

preparations (data not shown). PNP was used as an alternative substrate inhibitor of HCFC-132b metabolism by cytochrome P-450 IIE1, because PNP is preferentially metabolized by this isoform (16). PNP (300 pM) completely inhibited the metabolism of HCFC-132b by microsomes from both control and pyridine-induced rats (data not shown). HCFC-132b (5 mM) did not inhibit PNP metabolism by microsomes from control rats and inhibited PNP metabolism by pyridineinduced microsomes by approximately 15% (data not shown).

Discussion The metabolic pathway for HCFC-132b elucidated in the present study is presented in Figure 8. The results indicate that HCFC-132b (1) is metabolized by hydroxylation to afford the geminal halohydrin 1,2-dichloro-2,2difluoroethanol (2), which may lose HC1 to yield chlorodifluoroacetaldehyde (3). Solvation of 3 gives chlorodifluoroacetaldehydehydrate (4). The analogue trichloroacetaldehyde (chloral) is also fully hydrated in water (22). The observation that chlorodifluoroacetaldehyde hydrate was the only fluorinated product of the in vitro microsomal metabolism of HCFC-132b supports the hypothesis that this aldehyde is the first stable product expected after cytochrome P-450 catalyzed hydroxylation of HCFC-132b (4). Chlorodifluoroacetaldehyde may undergo reduction (5), which is metabolized to 2-chloro-2,2-difluoroethanol to an unidentified conjugate 6 (metabolite A) and to glu-

curonide 7, or may undergo oxidation to chlorodifluoroacetic acid (8). Alternatively, the aldehyde hydrate may be converted to a hemiacetal 9 (metabolite B) by conjugation to an unknown moiety. Reduction of the intermediate chlorodifluoroacetaldehyde to 2-chloro-2,2-difluoroethanol followed by glucuronide formation is the predominant initial pathway of metabolism, whereas oxidation to chlorodifluoroacetic acid predominates after repeated exposure to HCFC-132b. The metabolic scheme shown in Figure 8 accounts for the failure to detect chlorodifluoroacetylated liver proteins: no acylating intermediate is formed during the metabolism of HCFC-132b. The formation of such intermediates occurs during the metabolism of compounds containing geminal dihalomethyl groups (CHX2), such as 2,2-dichloro-l,l,l-trifluoroethane(HCFC-123) and halothane (19) (Figure 1B). The scheme presented in Figure 8 is consistent with the fate of the analogue 1,1,1,2-tetrachloroethane: 2,2,2-trichloroethanol,2,2,2-trichloroethyl glucuronide, and trichloroacetate were identified as urinary metabolites in several species (23,24). The complexity of the 19FNMR spectrum of 2-chloro2,2-difluoroethyl glucuronide, which is characterized by two 3JHF= 11.3 Hz triplets separated by 4 Hz (Figures 2 and 3), can be explained by second-order spectral interactions. Proton NMR spectra of glucuronides bearing aglycons with C-1 hydrogens, including 2,2,2-trifluoroethyl glucuronide, also show second-order spectral effects (21). Conformationallycontrolled long-range coupling interactions were ruled out as a cause of this phenomenon. In support of this conclusion, the 19FNMR spectrum of 2chloro-2,2-difluoroethylglucuronide was collapsed to a 5-Hz doublet by broad-band lH decoupling (data not shown). Efforts to identify the structures of metabolites A and B are in progress. A hemiacetal conjugate of an aldehyde hydrate (e.g., metabolite B) has apparently not been previously identified, although the formation of such metabolites has been suggested (25). Because cytochrome P-450 isoform IIEl catalyzes the metabolism of HCFC-134a (26)and halothane (27),its role in HCFC-132b metabolism was investigated. Cytochrome P-450 IIEl activities are induced by pyridine treatment (15). HCFC-132b metabolism was increased in microsomes from pyridine-treated rats (Figure 7), indicating the in-

Chem. Res. Toxicol., Vol. 4, No. 2, 1991 185

Metabolism of HCFC-132b

‘Acl , P-450

CI

u“;-.3- HCI

+

H20

___)

c1

OH

2

-

F

3

I

H6

3, chlorodiFigure 8. Proposed pathway of HCFC-132b metabolism in the rat. 1, HCFC-132b; 2, 1,2-dichloro-2,2-difluoroethanol; 6, metabolite A; 7,2-chloro-2,2-difluoroethyl fluoroacetaldehyde; 4, chlorodifluoroacetaldehydehydrate; 5,2-chloro-2,2-difluoroethano~ glucuronide;8, chlorodifluoroaceticacid; 9, metabolite B. Metabolites shown in brackets have not been characterized.

volvement of cytochrome P-450 IIEl in HCFC-132b metabolism. Moreover, PNP, a selective substrate for cytochrome P-450 IIE1, inhibited the microsomal metabolism of HCFC-132b. (HCFC-132b produced little inhibition of P N P metabolism. The failure of HCFC-132b to inhibit P N P metabolism is not due to limitations in solubility, because HCFC-132b is soluble in water to at least 20 mM). Olson et al. showed that P N P is a competitive inhibitor of HCFC-134a defluorination, with an apparent Ki of 36 pM (26). Pyridine treatment of rats increased the rate of HCFC-134a metabolism by hepatic microsomes (26). Furthermore, of several purified cytochrome P-450 isoforms studied, IIEl was the most active in HCFC-134a defluorination (26). These data indicate that cytochrome P-450 isoform IIEl may play a major role in HCFC metabolism, although a role for other isoforms is not excluded by these findings. Cytochrome P-450 IIEl is present in human liver (P-450 HLj) and is induced by isoniazid treatment and by alcohol consumption (28,29), indicating that humans may also metabolize HCFCs. Repeated exposure of rats to HCFC-132b increased both the relative fraction of the HCFC-132b dose excreted as chlorodifluoroacetic acid and the rate of chlorodifluoroacetic acid excretion (Figure 6, panels A and B, respectively). This observation indicates that one or more of the enzymes catalyzing aldehyde oxidation may be induced by repeated exposure to HCFC-132b. Aldehydes are oxidized to acids by aldehyde dehydrogenases, aldehyde oxidase, xanthine oxidase (30), and cytochrome P-450 (31). The metabolism of trifluoroacetaldehyde hydrate to trifluoroacetic acid is apparently not catalyzed by aldehyde dehydrogenases or aldehyde oxidase, but the enzymes responsible have not been identified (32). HCFC-132b has demonstrated toxicity (1l),whereas the fully fluorinated analogue HCFC-134a is relatively nontoxic (6). The observed toxicities of HCFC-132b and HCFC-134a parallel the extent of their metabolism, indicating an association of HCFC metabolism with toxicity.

Approximately 2 % of a single dose of HCFC-132b (10 mmol/kg, ip) is excreted as polar urinary metabolites during the first 24 h. HCFC-l34a, the fully fluorinated analogue of HCFC-l32b, is not extensively metabolized, however, either in vitro (9,10) or in It is not known whether the observed toxicity of HCFC-132b (11) is associated with its metabolism to 2-chloro-2,2-difluoroethanol, but the analogue 2,2,2-trifluoroethanol is toxic in rats (33). Comparison of the rates of metabolism of HCFC-132b and HCFC-134a indicates that the rates of 1,1,1,2-tetrahaloethane metabolism are controlled by the degree of fluorination. Further studies of substituent effects, in particular, the degree and placement of chlorine, on HCFC metabolism may reveal generalizationsabout the structural factors that control rates of metabolism and may aid in the design of candidate HCFCs that are resistant to metabolism. Acknowledgment. We thank M. J. Olson, General Motors Research Laboratories, for helpful discussions and S. E. Morgan for assistance in the production of the figures. J.W.H. was supported by National Institute of Environmental Health Sciences Training Grant ES07026. This research was supported in part by National Institute of Environmental Health Sciences Grant ES05407 to M.W.A. Note Added in Proof. Metabolite A has been identified as 2-chloro-2,2-difluoroethylsulfate. The sulfate ester was prepared by reaction of pure, dry 2-chloro-2,2-difluoroethanol with chlorosulfonicacid and was purified by XAD chromatography. The product was characterized by FAB mass spectrometry; synthetic 2-chloro-2,2-difluoroethyl sulfate coresonated with metabolite A (either purified from rat urine by XAD chromatography, anion-exchange chromatography, and reversed-phase HPLC or present in the :’ J. W.

Harris, M. J. Olson, and M. W. Anders, unpublished results.

186 Chem. Res. Toxicol., Vol. 4, No. 2, 1991

untreated urine of rats exposed to HCFC-l32b), and both showed identical I9Fspectral values (Table I). Synthetic 2-chloro-2,2-difluoroethylsulfate dissolved in 0.15 M acetate buffer, pH 5.0, was readily cleaved by incubation at 37 "C with sulfatase; 2-chloro-2,2-difluoroethylsulfate purified from the urine of HCFC-132b-treated rats was similarly hydrolyzed. When 2-chloro-2,2-difluoroethyl sulfate was added to control rat urine, however, little sulfatase-dependent hydrolysis was observed after incubation at 37 "C, pH 5.0. Apparently endogenous compounds present in urine prevent hydrolysis of the sulfate ester, which explains the failure to observe sulfatase-catalyzed cleavage of metabolite A in our original studies.

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