Chem. Res. Toxicol. 1993,6, 630-634
630
Slow-Binding Inhibition of Carboxylesterase and Other Serine Hydrolases by Chlorodifluoroacetaldehyde H e q u n Yin, Jeffrey P. Jones, and
M. W. Anders*
Department of Pharmacology, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642 Received March 5, 199P
The chlorofluorocarbon substitute 1,2-dichloro-l,l-difluoroethane (HCFC-132b) undergoes oxidative metabolism in rats to give a range of metabolites, including chlorodifluoroacetaldehyde [Harris and Anders (1991) Chem. Res. Toxicol. 4,180 3. The present experiments were undertaken after studies to characterize an unidentified metabolite of HCFC-132b revealed that chlorodifluoroacetaldehyde was toxic in vivo: rats given chlorodifluoroacetaldehyde died showing signs of cholinergic stimulation. Because some fluoroketones are known inhibitors of hydrolases, including acetylcholinesterase, the inhibitory effects of chlorodifluoroacetaldehyde on acetylcholinesterase (electric eel and human erythrocyte), on pseudocholinesterase (horse serum), on carboxylesterase (pig liver), and on a-chymotrypsin (bovine pancreas) were studied. In aqueous hydrate, as determined solution, the ratio chlorodifluoroacetaldehyde:chlorodifluoroacetaldehyde by ‘H nuclear magnetic resonance spectroscopy, was 1:157. Chlorodifluoroacetaldehyde was a slow-binding inhibitor of both acetylcholinesterases, of pseudocholinesterase, and of carboxylesterase; the Ki values, corrected for the aldehyde:hydrate ratio, were 150 nM, 1.7 nM, 3.7 nM, and 23 pM, respectively, as determined by final velocity of the progress curves; the k, values were 9.1 X lo4, 1.1 X lo6, 3.2 X lo4,and 9.2 X los M-l min-l, respectively. Chlorodifluoroacetaldehyde did not inhibit a-chymotrypsin. Acetaldehyde and trichloroacetaldehydewere classical competitive inhibitors of acetylcholinesterase. These results show that hydrochlorofluorocarbon metabolites may exert significant biological effects. Introduction
Hydrochlorofluorocarbons and hydrofluorocarbons are being developed as replacements for chlorofluorocarbons that lower stratospheric ozone concentrations (1,2). The presence of C-H bonds in hydrochlorofluorocarbons renders them tropospherically labile and prevents their migration to the stratosphere. Similarly, the presence of C-H bonds in hydrochlorofluorocarbons may be expected to make them metabolically labile (3). Indeed, recent (HCFCstudies show that 2,2-dichlorc~l,l,l-trifluoroethane 123) (4,5),2-chloro-1,1,1,Btetrafluoroethane (HCFC-124) (51, pentafluoroethane (HFC-125) (51, 1,1,1,24etrafluoroethane (HFC-134a) (6-10), 1,l-dichloro-l-fluoroethane (HCFC-141b) (1I), and 1,Bdichloro-1,l-difluoroethane (HCFC-132b)l (12) all undergo oxidative metabolism. Although the toxicity of most hydrochlorofluorocarbons and hydrofluorocarbons has not been fully studied, rats a Abstract
published in Advance ACS Abstracts, August 15, 1993. HCFC-l32b, 1,2-dichloro-l,l-difluoroethane. 2 The uncharacterized ‘Metabolite B” reported by Harris and Anders (12) was identified as N-(l-hydroxy-2-chloro-2,2-difluoroethyl)urea (FZClCCH(OH)NHC(=O)NHz).The 19F NMR spectrum of rat urine incubated with chlorodifluoroacetaldehyde hydrate, of 0.1 M urea incubated with chlorodifluoroacetaldehydehydrate, or of the urine of rata given chlorodifluoroacetaldehydehydrate showed an AB spectral pattern identical with that seen in the urine of rats given 1,2-dichloro-1,l-difluoroethane:19F NMR (254.18 MHz, D 2 0 , 6 m - d e = 0): 6 8.19 (Vpp = 166 Hz, = 4.1 Hz), 9.06. When solutions containing N-(l-hydroxy-2-chloro-2,2-difluoroethyl)urea were heated in a boiling water bath, the resonances aesigned to the amino alcohol decreased in intensity and a doublet appeared downfield in the 19F NMR spectrum. These resonances were assigned to the iminium ion (F&lCCH= NH+C(=O)NH2)formed by dehydration of the amino alcohol: 19FNMR (254.18 MHz, DzO, 6 m w w &= 0): 6 11.36 (d, ~ J w 6.1 Hz); ‘H NMR (270.13 MHz, D?O, 6 - & , . ~ = 0): 6 5.29 (m, H)!5.76 ( 8 , 2 H), 6.69 (d, trans-iminium ion, J = 9.9 Hz), 7.00 (d, cis-immium ion, J = 5.8 Hz); mp = 108.5-110 OC. Selinsky et al. (30) have identified an analogous iminium ion in the urine of rata given 2,2,2-trifluoroethanol. 1 Abbreviation:
exposed for 90 days to HCFC-132b showed 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 (13). Previous work in this laboratory showed that HCFC132b undergoes cytochrome P-450-catalyzed oxidative metabolism in rata to give a range of metabolites (12).19F NMR studies of urine fromrats given HCFC-132b showed the presence of chlorodifluoroacetic acid, 2-chloro-2,2difluoroethyl glucuronide, 2-chloro-2,2-difluoroethylsulfate, chlorodifluoroacetaldehyde hydrate, and an unidentified metabolite derived from chlorodifluoroacetaldehyde. During the course of studies to characterize the unidentified, chlorodifluoroacetaldehyde-derivedmetabolite, rata were given chlorodifluoroacetaldehyde.2 Unexpectedly, the rats given chlorodifluoroacetaldehyde died showing signs of cholinergic stimulation. Because some trifluoromethyl ketones are inhibitors of hydrolases, including acetylcholinesterase (141,studies were undertaken to determine whether the observed toxicity of chlorodifluoroacetaldehyde may be associated with the inhibition of acetylcholinesterase. Chlorodifluoroacetaldehyde inhibited acetylcholinesterase,and the studies were extended to include examination of the inhibitory effects of chlorodifluoroacetaldehyde on a range of serine hydrolases. In addition to acetylcholinesterase, chlorodifluoroacetaldehyde inhibited pseudocholinesterase and carboxylesterase, but did not inhibit a-chymotrypsin. Experimental Procedures Materials. Acetylcholinesterase (EC 3.1.1.7) from electric eel (Sigma type 111) and from human erythrocytes (Sigma type
0893-228~/93/2706-0630$04.00/0 0 1993 American Chemical Society
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 631
Hydrolase Inhibition by Chlorodifluoroacetaldehyde XIII), pseudocholinesterase (EC 3.1.1.8) from horse serum, carboxylesterase (EC 3.1.1.1) from porcine liver, and a-chymotrypsin (EC 3.4.21.1) from bovine pancreas (Sigma type I) were purchased from Sigma Chemical Co. (St. Louis, MO) and were used without further purification. Ethyl chlorodifluoroacetate was purchased from PCR, Inc. (Gainesville, FL). Acetylthiocholine, butyrylthiocholine, p-nitrophenyl acetate, and N-benzoyl-L-tyrosine ethyl ester were also obtained from Sigma Chemical Co. All other organic and inorganic compounds were purchased from commercial sources. Instrumental Analyses. lgF NMR spectra were obtained with a Bruker WP-270 NMR spectrometer equipped with a 5-mm 19F probe and operating at 254.18 MHz. Spectra were acquired at room temperature with sample spinning. Chemicalshifts were referenced to a 5.0 mM solution of trifluoroacetamide in DzO (6 = 0) contained in a sealed coaxial tube. 1H NMR spectra were recorded with the same instrument operating at 270.13 MHz and equippedwith a 5-mm lH probe. 1H chemicalshifts are expressed in ppm downfield from tetramethylsilane. Syntheses. Chlorodifluoroacetaldehyde was obtained by synthesis (15).Chlorodifluoroacetaldehydereacted instantly with water to give chlorodifluoroacetaldehyde hydrate: lH NMR (DzO): 6 5.10 (t, 1H, JH-F = 4.0 Hz); '9F NMR (DzO): 6 6.38 (d, JH-F= 4.0 Hz). Enzyme Assays. Progress curves were measured at 30 'C in 10" path length cuvettes with a Beckman DU-65spectrometer equipped with a six-cell automatic sampler. The reaction mixtures contained 30 mM potassium phosphate buffer (pH 7.0), 0.05-2.5 mM chlorodifluoroacetaldehyde hydrate, enzyme, and substrate in a final volume of 1.0 mL; reactions were started by addition of enzyme. Electric eel acetylcholinesterase activity was measured with acetylthiocholine as the substrate (16);see legend to Figure 1for details. In other experiments, acetaldehyde (0, 1,5, or 10 mM) and trichloroacetaldehyde, added as chloral hydrate (0, 0.5, 2, or 5 mM), were used as the inhibitors; the substrate concentrations were 0.035-0.14 mM acetylthiocholine. Human erythrocyte acetylcholinesterase activity (0.01unit/mL) was determined with 0.014 mM acetylthiocholineas the substrate. Pseudocholinesterase activity (0.013 unit/mL) was determined with butyrylthiocholine (0.25 mM) as the substrate (16). Carboxylesterase activity was quantified withp-nitrophenyl acetate as the substrate (17); see legend to Figure 2 for details. dhymotrypsin activity was determined spectrophotometrically with N-benzoyl-L-tyrosineethyl ester as the substrate (18);see Results for details. Activities were estimated from the linear increase in absorbance per minute under conditions demonstrating first-order rate dependence on the enzyme assayed. Kinetic Analyses. Ki values were determined from the final velocities of progress curves, as described by Morrison et al. (19). The progress curves for the reaction of chlorodifluoroacetaldehyde with hydrolases fit best to a one-step mechanism for reversible, slow-binding inhibition, which is described by the integrated equation:
0.20
-.m
c
0
2
4
6
8
10
12
14
16
Time (min)
Figure 1. Progress curves for the reaction of electric eel acetylcholinesterase with acetylthiocholine. The reaction mixtures contained 30 mM potassium phosphate buffer (pH 7.0), 0.31 mM 5,5'-dithiobis(2-nitrobenzoic acid), 0.14 mM acetylthiocholine, 0.01 unit of acetylcholinesterase/mL,and 0 (O),0.05 (n),0.3 (A),or 1.0 ( 0 )mM chlorodifluoroacetaldehyde in a total volume of 1mL and were incubated at 30 OC. Enzyme activity was measured as described in Experimental Procedures. The solid lines were generated by fitting the data to eq 1. fluoroacetaldehyde hydrate ratio, presuming that the aldehyde is the actual inhibitor of the enzyme.
Results Determination of Chlorodifluoroacetaldehyde to ChlorodifluoroacetaldehydeHydrate Ratio. Addition of chlorodifluoroacetaldehyde t o water resulted in t h e immediate formation of chlorodifluoroacetaldehyde hydrate. T h e lH NMR spectrum of a mixture of chlorodifluoroacetaldehyde and chlorodifluoroacetaldehyde hydrate in DzO gave two resonances: 6 5.1 (t, JH-F = 4.0 Hz, C(OH)fi), 9.5 (s, C(=O)H). Integration of t h e two resonances gave a ch1orodifluoroacetaldehyde:chlorodifluoroacetaldehyde hydrate ratio of 1:157. Inhibition of Acetylcholinesterases. The inhibitory effect of chlorodifluoroacetaldehyde on electric eel acetylcholinesterase was studied (Figure 1). Chlorodifluoroacetaldehydewas a slow-binding inhibitor of acetylcholinesterase, as evidenced by the decrease in the rate of product formation with time (19). T h e Ki, determined from eq 1, was calculated from the final velocities of the progress curves and amounted to 150 nM (Table I). T h e association rate constant k,, was 9.1 X lo4
M-l min-l.
P = Vat + (V, - VJ(1- eAt)/k
(1) where P = product formation, Vu = steady-state velocity, VO= initial velocity, t = time, and k = apparent first-order rate constant, which was obtained by a nonlinear, least-squares fit to eq 1. k is a function of k0eand Ki,as expressed in the equation:
k = kofi[l + [II/(Ki(l+ S/K,))I
t
0.60
(2)
Ki and k o were ~ also obtained by a nonlinear, least-squares fit to eq 1. The Ki thus obtained is a true inhibition constant and is corrected for the decrease in free inhibitor concentration due to enzyme-inhibitor complex formation. The association rate constant k, was calculated from koff/Ki.Kinetic constants were computed by the EZ-FIT program (Perrella Scientific, Inc., Conyers, GA). Substrate concentrations used in data analyses are based on the concentration of added chlorodifluoroacetaldehyde, which was added as an aqueous solution. Calculated Ki values were corrected for chlorodifluoroacetaldehyde:chlorodi-
T h e effect of pH on t h e inhibitory action of chlorodifluoroacetaldehyde on electric eel acetylcholinesterase was examined. At pH = 7.4, the Ki = (2.8 f 0.4) X 10-7 M (mean f SD, N = 3),kon = (7.9 f 0.3)x lo4M-l min-1, and k,ff = (2.2 f 0.3) X These kinetic constants are in good agreement with the values obtained at pH 7.0 (Table
1). Trichloroacetaldehyde was investigated as potential inhibitor of acetylcholinesterase. Trichloroacetaldehyde, added as chloral hydrate, was a classical competitive inhibitor of electrical eel acetylcholinesterase, with Ki of 60 f 11 pM (mean f SD, N = 3). Acetaldehyde also inhibited electric eel acetylcholinesterase, but only at high concentrations. Acetaldehyde was a classic competitive inhibitor, a n d t h e Ki was 990 f 570 pM ( m e a n f SD, N = 3). T h e s e data s h o w t h a t
632 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
Yin et al.
Table I. Inhibition of Hydrolases by Chlorodifluoroacetaldehyde* enzyme Ki (M) k, (M-l min-1) k , (min-1) ~ acetylcholinesterase (electric eel) (1.5 f 1.1) x 10-7 (9.1 f 1.6) X lo" (1.3 f 0.7) X acetylcholinesterase (human erythrocyte) (1.7 f 1.0) x 10-9 (1.1 0.5) x 105 (1.6 f 0.4) X lo-' pseudocholinesterase (horse serum) (3.7f 0.2) x 10-9 (3.2f 0.4)X lo" (1.2f 0.2) x lo-' carboxylesterase (pig liver) (2.3f 2.0) X 10-" (9.2f 2.5) X 106 (2.4f 2.5) X 1W @-chymotrypsin(bovine pancreas) no inhibition no inhibition no inhibition a The indicated enzymes were incubated with substrates and chlorodifluoroacetaldehyde, as described in Experimental Procedures. Kinetic constants were calculated from eq 1 and are corrected for the ch1orodifluoroacetaldehyde:chlorodifluoroacetyl~dehyde hydrate ratio. Data are expressed as mean f SD, N = 3. 0.60~
aromatic amino acids as well as peptides and proteins. Trifluoromethyl ketones, which exist largely as the hydrate, are known inhibitors of chymotrypsin (21). Hence it was of interest to determine whether chlorodifluoroacetaldehyde inhibited a-chymotrypsin activity. Chlorodifluoroacetaldehyde (0.05-5 mM) failed to inhibit a-chymotrypsin (0.0075 pg of protein/mL) with N-benzoyl-Ltyrosine ethyl ester (0.3 mM) as the substrate.
0,401
Discussion
0.00
b 0
2
4
6
8
10
12
14
Time (mln)
Figure 2. Progress curves for the reaction of porcine liver carboxylesterase with p-nitrophenyl acetate. The reaction mixtures contained30 mMpotassiumphosphate buffer (pH 7.0), 0.40 mMp-nitrophenylacetate,0.01unit of acetylcholinesterase/ mM chlorodifluoromL, and 0 ( O ) , 0.05 (A), 0.3 (a),or 1.0 (0) acetaldehyde in a total volume of 1 mL and were incubated at 30 "C. Enzyme activity was measured as described in Experimental Procedures. The solid lines were generated by fitting the data to eq 1.
acetaldehyde is less potent as an inhibitor of acetylcholinesterase than are chlorodifluoroacetaldehydeand trichloroacetaldehyde, indicating that a-halogen substitution of acetaldehyde,especially fluorine substitution, potentiates its inhibitory effects. The inhibition of human erythrocyte acetylcholinesterase by chlorodifluoroacetaldehyde was also examined. Chlorodifluoroacetaldehyde was a slow-binding inhibitor of human erythrocyte acetylcholinesterasewith aKi of 1.7 nM (Table I). The association rate constant was 1.1 X lo5 M-1 min-1, which is comparable to the value obtained for electric eel acetylcholinesterase. Inhibition of Pseudocholinesterase. Chlorodifluoroacetaldehyde inhibited pseudocholinesterase with butyrylthiocholine as the substrate. Chlorodifluoroacetaldehyde was a slow-binding inhibitor of pseudocholinesterase, with a Ki value of 3.7 nM. The association rate constant was 3.2 X lo4 M-l min-l. Inhibition of Carboxylesterase. Carboxylesterases catalyze the hydrolysis of a variety of uncharged esters, including aromatic and aliphatic esters, thioesters, and amides (20). The inhibitory effects of chlorodifluoroacetaldehyde on carboxylesterase, with p-nitrophenyl acetate as the substrate, were investigated (Figure 2). Chlorodifluoroacetaldehydewas a slow-bindinginhibitor of pseudocholinesterase; the Ki value was 23 pM, and the association rate constant was 9.2 X lo5 M-' min-' (Table I). Inhibition of a-Chymotrypsin. a-Chymotrypsin catalyzes the hydrolysis of various esters and amides of
The observation that chlorodifluoroacetaldehyde was toxic in rats and produced signs of cholinergic stimulation raised the possibility that chlorodifluoroacetaldehyde inhibited acetylcholinesterase, which could account for the observed toxicity. Moreover, some fluoromethyl ketones inhibit hydrolases, including acetylcholinesterase (14, 22, 23). This possibility proved to be correct: preliminary studies showed that chlorodifluoroacetaldehyde inhibited electric eel acetylcholinesterase. The studies were extended to include a range of serine hydrolases, including electric eel and human erythrocyte acetylcholinesterase, pseudocholinesterase, carboxylesterase, and a-chymotrypsin. The objective was to characterize the kinetics of inhibition of hydrolases by chlorodifluoroacetaldehyde. In addition, the inhibitory effects of the congeners acetaldehyde and trichloroacetaldehye on acetylcholinesterase were studied. The rank order of inhibition was carboxylesterase >> human erythrocyte acetylcholinesterase = horse serum pseudocholinesterase >> electric eel acetylcholinesterase; chlorodifluoroacetaldehydedid not inhibit a-chymotrypsin (Table I). The association rate constants k, ranged from 3.2 X lo4 to 9.2 X lo5 M-l min-l for the hydrolases, and the dissociation rate constants k,ff ranged from 2.4 X 106 for carboxylesterase to 1.3 X min-l for electric eel acetylcholinesterase. Chlorodifluoroacetaldehyde was a potent inhibitor of carboxylesterase, which is the least selective hydrolase studied. Apparently, the active site of carboxylesterase imposes few restrictions on the substrate, and chlorodifluoroacetaldehyde can be readily accommodated. Acetylcholinesterase and pseudocholinesterase selectively hydrolyze positively-charged choline esters, and both enzymes possess an anionic site and an esteric site. Allen and Abeles (14) found that uncharged trifluoromethyl ketones inhibit acetylcholinesterase and that inhibition was not enhanced in the presence of tetramethylammonium chloride. The failure of chlorodifluoroacetaldehyde to inhibit a-chymotrypsin indicates that the binding affinity of chlorodifluoroacetaldehydefor a-chymotrypsin is low. Although the reason for the failure of chlorodifluoroacetaldehyde to bind strongly to a-chymotrypsin is unknown, it is likely that the poor binding arises from a-chymotrypsin's preference for large peptide substrates.
Hydrolase Inhibition by Chlorodifluoroacetaldehyde
Scheme I. Proposed Mechanism of Inhibition of Serine Hydrolases by Chlorodifluoroacetaldehyde
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 633
Grant ES05407. The authors thank Sandra E. Morgan for assistance in preparing the manuscript.
References (1) McFarland, M., and Kaye, J. (1992)Chlorofluorocarbonsand ozone.
The inhibition of serine hydrolases by fluoromethyl ketones results from the covalent reaction of the ketone carbonyl group and the serine hydroxyl group in the active site of the enzyme to give a tetrahedral intermediate that may serve as a transition-state inhibitor of the enzyme (14,22,23).By analogy, chlorodifluoroacetaldehyde may react with active-site serine residues in hydrolases to form hemiketals that may serve as transition-state inhibitors (Scheme I). Short-chain aldehydes are in equilibrium with their corresponding hydrates, and the aldehyde, rather than the hydrate, reacts at the active site. The effect of pH on the inhibition of acetylcholinesterase by chlorodifluoroacetaldehydewas studied. Although the activity of acetylcholinesterase is lower at physiological pH (pH 7.4) than at pH 7.0 (data not shown), the kinetic constants for inhibition by chlorodifluoroacetaldehyde were similar at both pH values. a-Halogen substitution, especiallyfluorine substitution, increases the electrophilic character of the aldehydic carbonyl group, thereby making it more reactive toward nucleophiles. Indeed, chlorodifluoroacetaldehyde was about ten thousand times more potent than acetaldehyde as an inhibitor of acetylcholinesterase, indicating that halogen substitution alters profoundly the reactivity of aldehydes with hydrolases. Chloral hydrate is a classical competitive inhibitor of the esterase activity of sheep liver cytoplasmic dehydrogenase (24),with Ki value of 42 pM (uncorrected for trichloroaceta1dehyde:chloral hydrate ratio). Although the data show that chloral hydrate inhibits anticholinesterase in vitro, inhibition of acetylcholinesterase by chloral hydrate in vivo has apparently not been reported. The toxicological significance of the potent inhibition of hydrolases, particularly by chlorodifluoroacetaldehyde, is not understood. Mammalian liver esterases play a role in drug and pesticide detoxification (25). For example, the insecticide malathion is detoxified in mammals by ester hydrolysis (26). Thus inhibition of carboxylesterase by chlorodifluoroacetaldehydemay render mammals more susceptible to the toxic actions of esters. A 90-day inhalation study showed that HCFC-132b caused significant liver and testicular damage, a decrease in the liver-to-body weight ratio, and decreases in brain and testes weights (13). A role for chlorodifluoroacetaldehyde in the observed toxicity of HCFC-132b has, however, not been established. It may be important to note that the trifluoroacetaldehyde (27)and trifluoroacetaldehyde precursors (28)are testicular toxins. Moreover, trifluoroacetaldehyde is a putative metabolite of 1,1,1,2tetrafluoroethane (HFC-134a) (IO),which causes benign testicular tumors (29). Hence studies to determine whether a relationship between testicular toxicity and the production of trihaloacetaldehydes as metabolites of hydrochlorofluorocarbons and hydrofluorocarbons are warranted. Acknowledgment. This research was supported by National Institute of Environmental Health Sciences
Photochem. Photobiol. 55,911-929. (2) Manzer, L. E. (1990) The CFC-ozone issue: Progress on the development of alternatives to CFCs. Science 249, 31-35. ( 3 ) Anders, M. W. (1991) M e t a b o l i s m a n d toxicity of hydrochlorofluorocarbons: Current knowledge and needs f& the future. Enuiron. Health Perspect. 96, 185-191. Harris, J. W., Pohl, L. R., Martin, J. L., and Anders, M. W. (1991) Tissue acylation by the chlorofluorocarbon substitute 2,Bdichlorol,l,l-trifluoroethane (HCFC-123). Proc. Natl. Acad. Sci. U.S.A. 88, 1407-1410. Harris, J. W., Jones, J. P., Martin, J. L., LaRosa, A. C., Olson, M. J., Pohl, L. R., and Anders, M. W. (1992) Pentahaloethane-based chlorofluorocarbonsubstitutes and halothane: Correlation of in vivo hepatic protein trifluoroacetylation and urinary trifluoroacetic acid excretion with calculated enthalpies of activation. Chem. Res. Toxicol. 5, 720-725. Olson, M. J., Kim, S. G., Reidy, C. A., Johnson, J. T., and Novak, R. F. (1991) Oxidation of 1,1,1,2-tetrafluoroethane(R-134a) in rat liver microsomes is catalyzed primarily by cytochrome P450IIEl. Drug Metab. Dispos. 19, 298-303. Olson, M. J., and Surbrook, S. E., Jr. (1991) Defluorination of the CFC-substitute 1,1,1,2-tetrafluoroethane:Comparison in human, rat and rabbit hepatic microsomes. Toxicol. Lett. 69, 89-99. Olson, M. J., Reidy, C. A., and Johnson, J. T. (1990) Defluorination of 1,1,1,2-tetrafluoroethane(R-134a)by ret hepatocytes. Biochem. Biophys. Res. Commun. 166,1390-1397. Olson, M. J., Reidy, C. A., Johnson, J. T., and Pederson, T. C. (1990) Oxidative defluorination of 1,1,1,2-tetrafluoroethane(R-134a) by rat liver microsomes. Drug Metab. Dispos. 18,992-998. Surbrook, S. E., Jr., and Olson, M. J. (1992) Dominant role of cytochrome P-450 2E1 in human hepatic microsomal oxidation of the CFC-substitute 1,1,1,2-tetrafluoroethane.DrugMetab. Dispos. 20,518-524. Harris, J. W., and Anders, M. W. (1991) In vivo metabolism of the (HCFC-14lb). hydrochlorofluorocarbon1,l-dichloro-1-fluoroethane Biochem. Pharmacol. 41, R13-Rl6. Harris, J. W., and Anders, M. W. (1991) Metabolism of the hydrochlorofluorocarbon 1,2-dichloro-l,l-difluoroethane.Chem. Res. Toxicol. 4, 180-186. Kelly, D. P., and Chiu, T. (1989) Ninety-day inhalation toxicity study in rata with hydrochlorofluorocarbon 132b. Toxicologist 9, 140. Allen, K. N., and Abeles, R. H. (1989) Inhibition kinetics of acetylcholinesterase with fluoromethyl ketones. Biochemistry 28, 8466-8473. Yamada, B., Campbell, R. W., and Vogl, 0. (1977) Haloaldehyde polymers. VII. Polymerization of chlorodifluoroacetaldehyde. J. Polym. Sci. 15,1123-1135. Ellman, G. L., Courtney, K. D., Anders, V., and Featherstone, R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95, Allen, K. N., and Abeles, R. H. (1989)Inhibition of pig liver esterase by trifluoromethyl ketones: Modulators of the catalytic reaction alter inhibition kinetics. Biochemistry 28, 135-140. Hummel, B. C. W. (1959) A modified spectrophotometric determination of chymotrypsin, trypsin, and thrombin. Can. J.Biochem. Physiol. 37, 1393-1399. Morrison, J. F. (1982) The slow-binding and slow, tight-binding inhibition of enzyme-catalyzed reactions. Trends Biochem. Sci. 7, 102-105. Ocken, P. R., and Levy M. (1969) Purification and properties of pig liver esterase. Arch. Biochem. Biophys. 136, 259-264, Liang, R.-C., and Abeles, R. H. (1987) Complex of a-chymotrypsin and N-acetyl-tleucyl-L-phenylalanyl trifluoromethylketone: Strue turalstudieswith NMRspectroscopy. Biochemistry26,7603-7608. Hammock, B. D., Wing, K. D., McLaughlin, J., Lovell, V. M., and Sparks, T. C. (1982) Trifluoromethylketones as possible transition state analog inhibitors of juvenile hormoneesterase. Pestic. Biochem. Physiol. 17, 76-88. Linderman, R. J., Leazer, J., Roe, R. M., Venkatesh, K., Selinsky, B. S., and London, R. E. (1988) 19F-NMR Spectral evidence that 3-octylthio-l,l,l-trifluoropropan-2-one, a potent inhibitor of insect juvenile hormone esterase, functions as a transition state analog inhibitor of acetylcholinesterase. Pestic. Biochem. Physiol. 31,187194.
634 Chem. Res. Toxicol., Vol. 6,No. 5, 1993 (24) Kitaon, T.M.(1986)Effects of diethylstilbestrol, 2,2'-dithiodipyridine, and chloral hydrate on the esterase activity of sheep liver cytoplasmicaldehydedehydrogenase. Biochemistry 25,4718-4724.
(25)Mentlein,R.,andHeymann,E. (1984)Hydrolyeisofester-andamidetype drugs by the purifedisoenzymea of nonspecific carboxyleaterase from rat liver. Biochem. Pharmacol. 33, 1243-1249. (26)O'Brien,R. D. (1967)Ineecticides. Actionand Metabolism, pp 263264,Academic Press, New York. (27)Crank, G., Harding, D. R. K., and Szinai,S. S. (1970)PerfluoroaLkyl carbonylcompounds. 1. Perfluoroaldehydeand perfluorocarboxylic acid derivatives. J.Med. Chem. 13,1212-1215.
Yin et al. (28)Lloyd, S. C., Blackburn, D. M., and Foster, P. M. D. (1988) Triiuoroethanol and ita oxidative metabolites: Comparison of in vivo and in vitro effects in rat testis. Toricol. Appl. Pharmacol. 92,
390-401. (29) Zurer,P. (1992)CFC substitutescausee benigntumoreinrata. Chem. Eng. News 70 (38),6. (30)Selinsky, B. S.,Rusyniak, D. E., Wareheski, J. O., and Joseph, A. P. (1991)1SFNuclearmagneticreeonanceanalyeisoftrifluoroethanol metabolites in the urine of the Sprague-Dawley rat. Biochem. Pharmacol. 42,222S2238.
