The anaerobic dechlorination of trichlorofluoro-methane by rat liver preparations in vitro

Chem.-Biol. Interactions, 21 (1978) 277--288

277

© Elsevier/North-Holland Scientific Publishers Ltd.

THE ANAEROBIC DECHLORINATION OF TRICHLOROFLUOROMETHANE BY RAT LIVER PREPARATIONS IN VITRO

C. R O L A N D W O L F , L A U R E N C E J. K I N G and D E N N I S V. P A R K E Department of Biochemistry, University of Surrey, Guildford, Surrey, G U 2 5 X H (United Kingdom) (Received December 1st,1977) (Accepted February 11th, 1978)

SUMMARY

Incubation of trichlorofluoromethane with a liver microsomal fraction and an NADPH generating system under anaerobic conditions produced a metabolite dichlorofluoromethane, characterised by gas chromatography and mass spectrometry. The metabolic reaction was carried out by liver microsomes from the mouse, rabbit, hamster and rat and was increased by phenobarbitone pre-treatment. The formation of dichlorofluoromethane in vitro was enhanced by the addition of FMN, but partially inhibited by the presence of air, oxygen, SK&F 525-A, metyrapone and carbon tetrachloride and totally inhibited by carbon monoxide. The consumption of NADPH in the reaction was greater than could be accounted for by the production of dichlorofluoromethane indicating the possible formation of other metabolic products. It is suggested that trichlorofluoromethane interacts with the reduced form of cytochrome P-450 at the oxygen binding site and a possible mechanism for its subsequent reductive dechlorination is proposed.

INTRODUCTION

Trichlorofluoromethane is widely used as a major component of aerosol propellants. In contrast to the analogous compound carbon tetrachloride, there is no evidence that trichlorofluoromethane is hepatotoxic [1--3]. The toxicity of carbon tetrachloride is due to the formation of active, metabolic intermediates which may be absent or produced in much lower concentrations after trichlorofluoromethane administration [4]. Slater [4] explained this by the increased stability of the C-C1 bond in trichlorofluoromethane, due to the presence of the fluorine atom, resulting in biochemical stability. Reiner et al. [5] reported that carbon tetrachloride is converted to Abbreviation: g.l.c.,gas-liquidchromatography.

278 chloroform under anaerobic conditions b y rabbit liver microsomal fractions fortified with NADPH. We have shown that trichlorofluoromethane interacts with components of the liver microsomal mixed function oxidase system [6] and this work has been extended to demonstrate that trichlorofluoromethane is metabolised under anaerobic conditions. A preliminary report of this work has been published [7]. MATERIALS AND METHODS

Materials Trichlorofluoromethane and dichlorofluoromethane were obtained from Cambrian Chemicals Ltd., Croydon, Surrey. No halogenated impurities were detected using the g.l.c, analysis described. NADP ÷, NADPH, glucose-6phosphate and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co., London. All other chemicals were of reagent grade, obtained from commercially available sources. Animals Male Wistar albino rats, Porton strain (250--300 g), male Syrian golden hamsters (130--150 g), male Dutch Cross rabbits {3--3.2 kg) and male CF~ mice (20--25 g), supplied b y the University of Surrey Animal Unit, were allowed free access to food and water. Rats pre-treated with sodium phenobarbitone received 80 mg/kg b o d y weight dissolved in 0.9% saline, intraperitoneally, daily for 3 days prior to use. Controls received an equal volume of 0.9% saline. Preparation o f liver subcellular fractions Animals were killed b y cervical dislocation, the livers removed and rinsed in ice-cold 0.2 M potassium phosphate buffer, pH 7.4, containing 0.25 M sucrose. The livers were blotted dry, weighed and homogenized in 4 volumes of ice-cold 0.2 M potassium phosphate buffer, pH 7.4, containing 0.25 M sucrose using three return strokes of a Potter-Elvehjem, teflon-glass homogeniser. Subcellular fractions were obtained by differential centrifugation at 4°C (MSE High Speed 18, 8 X 50 ml rotor and MSE Superspeed 50, 8 X 25 ml rotor) essentially using the method of Amar-Costesec et al. [8]. In experiments requiring only the microsomal fraction, the liver homogenate was centrifuged at 15 000 gay for 20 min and the resulting supernatant centrifuged at 105 000 gay for 1 h. The microsomal pellet was resuspended in 0.2 M potassium phosphate buffer, pH 7.4. Protein determinations were made by the m e t h o d of Lowry et al. [9] using a bovine serum albumin standard. Cytochrome P-450 was determined using the method of Omura and Sato [10] using a Perkin Elmer, model 356 spectrophotometer. Incubation procedure Typical incubation experiments c o n t a i n e d : t i s s u e fraction (4 or 8 mg

279 protein/ml), NADP* (1.5 mM), glucose-6-phosphate (3.0 mM), glucose 6-phosphate dehydrogenase (2 units), trichlorofluoromethane or carbon tetrachloride (10 mM). Incubations were carried out in 0.2 M potassium phosphate buffer, pH 7.4, at a final volume of 10 ml in two-necked 25 ml flasks made anaerobic by 10-fold evacuation and flushing with nitrogen using a 3-way tap fitted into one of the necks. Substrate, trichlorofluoromethane or carbon tetrachloride, was injected into the incubation medium through a self-sealing stopper on the other side-arm, using a pre-cooled 10 pl g.l.c, syringe. The flasks were incubated at 37°C, with shaking (100 rev./min) for periods up to 1 h. The reaction was stopped by cooling the flasks in ice. The incubations were extracted with n-heptane (5 ml) and the extracts analysed by g.l.c. Trichlorofluoromethane was not lost from the apparatus during the incubation and the extraction of trichlorofluoromethane from the incubation medium was approx. 100%. Experiments were performed in which the following parameters were varied :time of incubation, microsomal protein concentration, NAD1~ concentration, in the presence of air, oxygen or carbon monoxide (a gas cylinder of one of these compounds was substituted for the nitrogen cylinder during the flushing procedure), sodium dithionite (2 mM), FMN (2 mM), SK&F 525-A (4 m M ) o r metyrapone (2 mM).

Gas-liquid chromatography All extracts were kept at 4°C in glass-stoppered flasks before analysis. The procedure adopted was a modified version of that reported by Cox [11]. A Pye-Unicam gas chromatograph, model 104, fitted with a 63Ni electron capture detector was used. The column was 15% squalane on a celite solid support, 80--120 mesh, in copper tubing, diameter 0.32 cm × 2 m. The operating conditions were: nitrogen flow rate, 60 ml/min; oven temperature, 60°C; detector pulse space, 150s; backing off range, × 100; injection volume, 5 ~1. The retention times obtained were: CHC12F, 2 min; CCI3F, 3 min; CHC13, 6.9 min and CC14, 13.1 m i n . Quantitation was obtained by plotting peak area against concentration of freshly-prepared standard solutions. Mass spectra Mass spectra were obtained from an LKB model 2091 mass spectrometer linked to a Pye-Unicam gas-liquid chromatograph fitted with the column described previously. To permit determination of mass spectra pooled n-heptane extracts from 10 microsomal incubations were distilled to give 3 ml of distillate and aiiquots (10 ~1) were injected into the gas chromatograph. Measurement o f trichlorofluoromethane-induced NADPH oxidation under anaerobic conditions NADPH oxidation was monitored in rat liver microsomal suspensions (2 mg protein/ml) from phenobarbitone pre-treated rats, in 0.066 M Tris--

280

HC1 buffer, pH 7.4, made anaerobic b y bubbling the suspension for 5 min at 4°C with oxygen-free nitrogen. The rate of decrease in the NADPH absorption at 340 nm was monitored at 37°C in stoppered 1 cm glass cuvettes in a Perkin Elmer spectrophotometer, model 356. Addition of trichlorofluoromethane (4 mM) in DMF was made immediately prior to the addition of N A D P H (66 pM). The rate of NADPH oxidation was measured in the absence of substrate (endogenous rate) and its presence (induced rate) in alternate samples. Absolute values were obtained with an extinction coefficient for N A D P H or 6.2 mM-]cm -' [12] using the trace obtained after the initial 30 s of reaction. The effect of carbon m o n o x i d e on the rate of NADPH oxidation in the presence of trichlorofluoromethane was determined b y bubbling the anaerobic microsomal suspension for 30 s with carbon m o n o x i d e before the addition of substrate and NADPH. RESULTS

G.l.c. analysis of extracts from 1 h anaerobic incubation of rat whole liver CCI F CHCI2F Standard solution Air

n-Heptane extract CCl3F

Metabolite

I

I

3

2

I

1 Retention time (min)

I

0

Fig. 1. Gas-liquid chromatographic analysis of an n-heptane extract of whole liver homogenate after anaerobic incubation with trichlorofluoromethane. The upper trace was that obtained from a standard solution o f CCI~F and CHCI2F. The lower trace was obtained from the n-heptane extract o f an anaerobic incubation (60 min) containing whole liver homogenate (2 g), NADPH generating system and CCI3F (10 mM). The g.l.c, operating conditions were as described in the Methods.

281

homogenate (2 g) with trichlorofluoromethane gave a peak with a retention time of 2 min identical to that of dichlorofluoromethane (Fig. 1). The peak was n o t detectable in incubations omitting tissue, NADP + or trichlorofluoromethane. Using carbon tetrachloride as substrate under identical conditions chloroform was detected, in agreement with Reiner et al. [5]. In 20 min incubations of trichlorofluoromethane with different subcellular fractions, 75% of the metabolic activity was found in the crude microsomal fraction. Mass spectral analysis of the g.l.c, peak of the metabolite is shown in Fig. 2 in comparison with that of an authentic sample of dichlorofluoromethane. The spectra are identical and show mass ions n o t present in a background scan. The assignments o f the major ions are: 102 (mol./ion), CH3SC12F*; 101, C3sC12F+; 82, C3SC12+; 67, CH3SC1F+; 66 (base peak), C3SC1F*; 48, CH3SCI+; 47

CHCI=F

l

m/e

I 30

I 40

10,

I 50

I 60

II

I 70

I 80

I,I

I 90

I,,

I 100

1 110

Metabolite

m/e

ii

,

30

40

il,, 50

, 60

I

l,

70

, [;I 80

, 90

, hi 100

, 110

Fig. 2. Mass spectra of metabolite obtained from trichlorofluoromethane and authentic dichlorofluoromethane. The sample of metabolite was obtained by distillation of pooled

n-heptane extracts of 10 anaerobic microsomal incubations carried out as described in the text. Microsomal protein concentration, 8 mg/ml. Incubation time, 40 rain.

282 CascI÷; 31, CF +. The corresponding peaks for aTC1 can be seen for the major ions. The mass spectrum differs considerably from that of the other major g.l.c, peak, trichlorofluoromethane, which has the base peak at 101 (C3sC12F+). The rate of formation o f dichlorofluoremethane b y rat liver microsomal fractions (Fig. 3) was linear for approximately 10 min and continued for up to 40 min. Variation was found between different microsomal preparations, b u t samples from the same preparation gave reproducible results. The enzymic nature of the reaction was supported by the observation of a linear relationship between the initial rate of formation of dichlorofluoromethane and microsomal protein concentration from 1--30 mg protein/ml. This enzymic activity was detected in the liver microsomal fractions of several species, the hamster having the highest activity in those investigated (Table I). Increasing the trichlorofluoromethane concentration increased the initial rate of formation of metabolite and high concentrations (20--30 mM) were required for maximum activity. This could be due to the low solubility of trichlorofluoromethane (b.p. 24°C) in the incubation medium at 37°C. Lineweaver-Burk plots of the reciprocal of the initial rate of formation of dichlorofluoromethane against reciprocal of substrate concentration (Fig. 4) gave V, 0.13 nmol/min/mg protein and apparent K m 1.9 mM. The true Km m a y be considerably less as this value was determined assuming that all the substrate was dissolved in the incubation medium. 6O

4A "o 3LK "I"

2-

I

I

10

20

I

I

J

I

30 40 50 Time (min) Fig. 3. R a t e o f f o r m a t i o n o f d i e h l o r o f l u o r o m e t h a n e b y a n a e r o b i c m i c r o s o m a l f r a c t i o n . I n c u b a t i o n c o n d i t i o n s w e r e as d e s c r i b e d in t h e t e x t . M i c r o s o m a l p r o t e i n c o n c e n t r a t i o n , 8 m g / m l . CC|3F c o n c e n t r a t i o n , 10 raM.

283 TABLE I THE FORMATION OF DICHLOROFLUOROMETHANE FROM TRICHLOROFLUOROMETHANE BY ANAEROBIC MICROSOMAL PREPARATIONS FROM SEVERAL SPECIES. The incubation conditions were as described in the text. Microsomal protein concentration, 8 mg/ml. CCI3F concentration, 10 raM. Incubation time, 20 rain.

Rat Rabbit Mouse Hamster

Number of experiments

Rate of formation of CHCI2F (nmol/min/mg protein)

10 3 4 3

0.044 0.040 0.043 0.055

The addition of sodium dithionite to the anaerobic incubation increased the yield of dichlorofluoromethane by approximately 30% but in the absence of the NADPH generating system only trace amounts were detectable. The addition of FMN (2 mM) produced a 50% increase but no production of metabolite in the absence of tissue. The addition of other compounds known to interact with the hepatic mixed function oxidase system decreased, or apparently decreased, the amount of dichlorofluoromethane produced in a 20 min incubation (Table II). Pretreatment of rats with phenobarbitone produced a 70% increase in the yield of dichlorofluoromethane in microsomal incubations which was closely paralleled by the increase in cytochrome P-450 (Table III). A small endogenous rate of NADPH oxidation (0.4 nmol/min/mg protein) was obtained in anaerobic incubations of liver microsomal preparations from phenobarbitone pre-treated rats (Fig. 5). Addition of trichlorofluoromethane in DMF increased the rate of oxidation to 1.6 nmol/min/mg protein while

Ai

2,0-

$

"D LL

1,0-

~O

-r

--0.6

--0.4

-0.2

0 0.2 0.4 I/[CCI3F ] (mM -z )

0.6

0.8

1.0

Fig. 4. Lineweaver-Burk plot of the effect of trichlorofluoromethane concentration on the formation of dichlorofluoromethane. Incubation proteins were as described in the text. Microsomal protein, 8 mg/ml. Incubation time, 20 min.

284 TABLE II THE E F F E C T OF VARIOUS COMPOUNDS ON THE FO RMA TI O N OF DICHLOROF L U O R O M E T H A N E FROM T R I C H L O R O F L U O R O M E T H A N E BY ANAEROBIC MICROSOMAL PREPARATIONS The results are expressed as the percentage inhibition of the dichlorofluoromethane production in control incubates. The incubation conditions were as described in the text. Microsomal protein concentration, 4 mg/ml. CC13F concentration, 10 raM. Incubation time, 20 rain.

Carbon monoxide (101 K.Pa) Oxygen (101 K.Pa) Air (101 K.Pa) SK&F 525-A (4 raM) Metyrapone (2 mM) Carbon tetrachloride (5 raM)

Number of experiments

Inhibition (%)

6 6 6 3 3 3

100 75 50 50 70 80

the addition of solvent alone was w i t h o u t effect. Carbon m o n o x i d e totally inhibited the trichlorofluoromethane-induced rate. DISCUSSION

The microsomal fraction of the livers of several species examined is capable of converting trichlorofluoromethane to dichlorofluoromethane under anaerobic conditions in the analogous manner to the reported production of chloroform from carbon tetrachloride [5,13]. The characteristics of the reaction suggest that it is enzymic and that it is mediated b y a component of the microsomal mixed function oxidase system. Preliminary inhibitor studies (Table II) and the parallel increase in diTABLE III THE E F F E C T OF PHENOBARBITONE PRE-TREATMENT ON THE PRODUCTION OF D I C H L O R O F L U O R O M E T H A N E FROM T R I C H L O R O F L U O R O M E T H A N E BY R AT LI VE R MICROSOMAL PREPARATIONS The incubation conditions were as described in the text using a microsomal protein concentration of 8 mg/ml, CCI3F (10 raM) and 20 rain incubation time. The values are mean ± S.E.M. for 10 experiments.

CHCI2F production (~g/min) Cytochrome P-450 (nmol/mg protein) ap<

0.05, b p < 0.01.

Control animals

Pretreated animals

% increase

0.33 ± 0.01

0.56 -+ 0.08 a

70

0.6

1.1

83

± 0.1

± 0.1 b

285 0.10 CCI3F + CO + NADPH NADPH

0.06 E

c O tO

CClsF + NADPH

ol 0.04W

0.02-

0

I

I

I

I

l

1

2

3

4

5

Time (min)

Fig. 5. Effect of trichlorofluoromethane and carbon monoxide on the anaerobic NADPH oxidation by phenobarbitone pre-treated rat liver microsomal preparations. The plots show the change in extinction at 340 nm. Incubation conditions were as described in the text. Microsomal protein concentration, 2 mg/ml. NADPH concentration, 66 uM. CClsF concentration, 4 raM.

chlorofluoromethane production with cytochrome P-450 induction in phenobarbitone pre-treated rats (Table III) suggest that the reaction takes place at a site on the cytochrome. Previous work has shown that trichlorofluoromethane binds to cytochrome P-450 under anaerobic and reducing conditions to produce a difference s p e c t r u m with a peak at 452 nm and also that trichlorofluoromethane reduces the magnitude o f the absorption peak at 450 nm of the carbon monoxide-cytochrome P-450 complex [6,7]. Oxygen binds to the c y t o c h r o m e at the same site as the carbon monoxide and also inhibits the production of dichlorofluoromethane from trichlorofluoromethane. It has been claimed that metyrapone [14] and SKF 525-A [15] which b o t h inhibit the reaction also bind to cytochrome P-450 under anaerobic conditions. All of this evidence points to the binding of trichlorofluoromethane at the oxygen site on the haem moiety of cytochrome P-450 and it is suggested that reductive dechlorination occurs by trichlorofluoromethane acting as the electron acceptor in the system in the absence of oxygen. This possibility can be incorporated into a scheme, similar to that proposed by Estabrook et al. [16] for the mechanism of mixed function oxidase metabolism as shown in Fig. 6. This mechanism proposes that various intermediate complexes exist with the cytochrome, namely the complete molecule (I), the free

286

P-450-Fe 2+

e- -¢,

I OCI sF

CCI 3F

CHCI2F 4 - - ~ !!

I //

CCI2F

J

I I I| l l Ii

--CCI2F

+

P-450-Fe2 (IV) _ I ~

P_450_Fe 2+ (II) I _ -- 'CCI2F //

~,~-450-Fe 3+

,

I

!

b5

,

i I I

(i)

P-450-Fe2 +

-- cyt

I

l I

e~

flavoprotein

NADPH

~.C1-

P-450-F 2+

(v) : CCL Fig. 6. P r o p o s e d m e c h a n i s m for t h e a n a e r o b i c m e t a b o l i s m o f t r i c h l o r o f l u o r o m e t h a n e b y t h e m i c r o s o m a l m i x e d f u n c t i o n o x i d a s e s y s t e m . T h e m e c h a n i s m is b a s e d u p o n t h e s c h e m e f o r o x i d a t i v e m e t a b o l i s m p r o p o s e d b y E s t a b r o o k e t al. [ 1 6 ] .

radical (II), the anions (III and IV) and the carbene (V). Evidence for the formation of carbene complexes, which account for the spectral interactions in the region of 450 nm, have been reported by Mansuy et al. [17] and Wolf et al. [18]. It is difficult to incorporate the carbene V, CC1F, as an intermediate in the production of dichlorofluoromethane and it is suggested that it m a y arise as an alternative in the breakdown of complex IV. Anaerobic microsomal incubation of trichlorofluoromethane with dithionite as the only reducing agent results in the time
287 the involvement of cytochrome P-450. Assuming that I molecule of NADPH is required for the metabolism of 1 molecule of trichlorofluoromethane only a small proportion of the intermediate complexes result in the formation of dichlorofluoromethane. Unpublished work (C.R. Wolf, T. Werner, H. Uekleke and L.J. King) has shown that under these experimental conditions metabolic products of 14CC13F bind to microsomal lipids and proteins at a rate which could account for this rate of NADPH oxidation. Complexes II--V contain reactive intermediates from trichlorofluoromethane which could result in covalent binding to tissue macromolecules. Under anaerobic conditions trichlorofluoromethane is not biologically inert and reacts in many respects like carbon tetrachloride. The inhibition of trichlorofluoromethane metabolism by carbon tetrachloride indicates that it has a lower affinity for cytochrome P-450. This together with the inhibition of this mode of metabolism by oxygen may partly explain why trichlorofluoromethane does not exhibit the hepatotoxicity of carbon tetrachloride. REFERENCES 1 J.W. Clayton, The mammalian toxicology or organic compounds containing fluorine, in O. Eichler, A. Farrah, H. Herken and A.D. Welch (Eds.), Handbuch der Experimentellen Pharmakologie, Springer, Berlin, Vol. 20, Part 1, 1966, p. 459. 2 J. Scholz, New toxicologic investigations of Freons used as propellants for aerosols and sprays, Berlin Aerosol-Kongress, 4 (1962) 420. 3 L.J. Jenkins, R.A. Jones, R.A. Coon and R. Siegel, Repeated and continuous exposures of laboratory animals to trichlorofluoromethane, Toxicol. Appl. Pharmacol., 16 (1970) 133. 4 T.F. Slater, A note on the relative toxic activities of tetrachloromethane and trichloro-fluoro-methane on the rat, Biochem. Pharmacol., 14 (1965) 178. 5 O. Reiner, S. Athanossopoulos, K.H. Hellmer, R.E. Murray and H. Uehleke, Formation of chloroform from carbon tetrachloride in liver microsomes, lipid peroxidation and destruction of cytochrome P-450. Arch. Toxicol., 29 (1972) 219. 6 P.J. Cox, L.J. King and D.V. Parke, The binding of trichlorofluoromethane and other haloalkanes to cytochrome P-450 under aerobic and anaerobic conditions, Xenobiotica, 6 (1976) 363. 7 C.R. Wolf, L.J. King and D.V. Parke, Anaerobic dechlorination of trichlorofluoromethane by liver microsomal preparations in vitro, Biochem. Soc. Trans., 3 (1975) 175. 8 A. Amar-Costesec, H. Beaufay, M. Wibo, D. Thin~s-Sempoux, E. Feytmans, M. Robbi and J. Berthet, Analytical study of microsomes and isolated subcellular membranes from rat liver, J. Cell Biol., 61 (1974) 201. 9 O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265. 10 T. Omura and R. Sato, The carbon monoxide-binding pigment of liver microsomes, J. Biol. Chem., 239 (1964) 2370. 11 P.J. Cox, Ph.D. thesis, University of Surrey, Guildford, Surrey, U.K., 1972. 12 H.U. Bergmeyer, H. Klotzsch, H. M611ering, M. Nelb~ck-Hochstetter and K. .Beaucamp, Biochemical reagents, in H.U. Bergmeyer (ed.), Methods of Enzymatic Analysis, Academic Press, New York and London, 1963, p. 1030. 13 H. Uehleke, K.H. Hellmer and S. Tabarelli, Binding of z4C-carbon tetrachloride to microsomal proteins in vitro and formation of CHC13 by reduced liver microsomes, Xenobiotica, 3 (1973) 1. 14 A.G. Hildebrandt, K.C. Leibman and R.W. Estabrook, Metyrapone interaction with

288

15 16 17 18

hepatic microsomal cytochrome P-450 from rats treated with phenobarbital, Biochem. Biophys. Res. Commun., 37 (1969) 477. J.B. Schenkman, B.J. Wilson and D.L. Cinti, Diethylaminoethyl 2,2-diphenylvalerate HCI (SKF 525-A)-in vivo and in vitro effects of metabolism by rat liver microsomesformation of and oxygenated complex, Biochem. Pharmacol., 21 (1972) 2373. R.W. Estabrook, A.G. Hilderbrandt, J. Baron, K.J. Netter and K. Leibman, A new spectral intermediate associated with cytochrome P-450 function in liver microsomes, Biochem. Biophys. Res. Commun., 42 (1971) 132. D. Mansuy, W. Nastainczyk and V. Ullrich, The mechanism of halothane binding to microsomal cytochrome P 4 5 0 , Arch. Pharmacol., 285 (1974) 315. C.R. Wolf, D. Mansuy, W. Nastalnczyk and V. Ullrich, The interaction of polyhalogenated methanes with ferrour cytochrome P-450., Biochem. Pharmacol.