Dechlorination ofhalocarbons by microsomes and vesicular

British Journal of Industrial Medicine 1985;42: 305-311

Dechlorination of halocarbons by microsomes andvesicular reconstituted cytochrome P-450 systemsunder reductive conditionsA G SALMON,' J A NASH,2 C M WALKLIN,3 AND R B FREEDMAN3

From the TUC Centenary Institute of Occupational Health, ' London School ofHygiene and TropicalMedicine, London WCIE 3HT, Central Toxicology Laboratory,2 Imperial Chemical Industries plc,Macclesfield, Cheshire, and Biological Laboratory,3 University ofKent, Canterbury, UK

ABSTRACr A spectrophotometric assay of the reductive dechlorination of halocarbons was

developed and used to determine the kinetic characteristics of dechlorination of a range ofhaloethanes catalysed by microsomes from rat and rabbit liver. Analysis of the typical reaction ofhexachloroethane shows that the reaction is catalysed by cytochrome P-450 and results in theformation of olefinic products as well as less chlorinated alkanes: in other respects the reactionresembles that known to occur with carbon tetrachloride. The dechlorination of haloethanescatalysed by a vesicular reconstituted system of cytochrome P-450 enzymes from rabbit liver wasalso studied and found to be similar to that catalysed by microsomes: both reductase and aphenobarbital inducible form of cytochrome P-450 were essential. There is no substantial depen-dence of maximum dechlorination rate on compound structure, suggesting that the reduction ofsubstrate is not the rate limiting step in the overall reaction. The main factor in determining theapparent binding constant to the enzyme is the partition coefficient into the lipid membrane, asassessed by calculated log P values.

The metabolism of halogenated hydrocarbonscommonly involves dehalogenation, which in manycases is dependent on microsomal cytochromeP-450. Van Dyke and Wineman showed that thedechlorination of chloroalkanes was dependent onNADPH and inhibited by CO; activity was alsoinducible by phenobarbital but not by3-methylcholanthrene.' Analysis of the reactivity ofhalocarbons as a function of their structure has beenattempted on the assumption of a single mechan-ism.2 We have shown previously, however, thenecessity of considering more than one mechanismin order to explain the behaviour of various com-pounds that have been studied in vitro.34 This is inagreement with the observations of products andmechanisms for those halocarbons that have beenstudied in detail: these data have been reviewedrecently.5 It seems that most dechlorination reac-tions are oxidative in character, involving an initialattack by an oxygenated complex of cytochrome

Received 20 August 1984Accepted 17 September 1984

P-450 on a hydrogen atom, resulting in rearrange-ment of the product with the loss of a halogen.Nevertheless, at least one other important mechan-ism of dechlorination exists, which is observed whenchemical structure or reaction conditions preventthe oxidative route. This mechanism, of which themetabolism of carbon tetrachloride is the bestknown example, involves reductive generation of aradical and direct release of a halide ion.The objectives of this study were: firstly, to define

a simple and reliable assay for the reductive reac-tion, based on the provision of a suitable anaerobicreaction medium.6 This needed to be sufficientlyreproducible to allow proper kinetic analysis of theresults. Secondly, to apply the assay and kineticanalysis to a range of haloethanes to investigate thestructure/reactivity relationship for the reductivereaction. Thirdly, to test the role of cytochromeP-450 isoenzymes and NADPH:cyt P-450 reductasedirectly by studies using reconstituted systems.Previous studies have reported reconstitution ofreductive dechlorination of carbon tetrachloride andhalothane in non-vesicular systems comprising

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cytochrome P-450, reductase, lipid, anddetergent.7 8To allow the use of the better defined and more

easily prepared rabbit P-450 enzymes, the reactionwas studied using both rabbit liver microsomes, withand without induction by phenobarbital andB3-naphthoflavone, and also the rat liver microsomesthat have been the subject of most previous work.These data were then compared with those obtainedwith purified cytochrome P-450 enzymes isolatedfrom rabbit liver in a reconstituted system of vesiclescontaining reductase, lipid, and cytochrome. Wehave investigated the progress of this reaction underfavourable conditions in vitro for a series of com-

pounds that were expected to be susceptible to thisprocess. Observed reactivities were correlated withstructural features of the reacting species. We havealso examined the metabolites formed in vitro froma typical compound, hexachloroethane. Furtherinvestigations performed with this compound were

designed to examine the effects of the mixed func-tion oxidase inhibitors carbon monoxide and SKF525A on the reaction.

Methods

PREPARATION OF MICROSOMESRat microsomes were prepared from the livers ofnormal (non-induced) male rats (Alderley Parkstrain, Wistar derived). The method of preparationhas been described.9 Food was withdrawn 24 hoursbefore death, and the washed microsomes were

stored under argon at -70°C. Rabbit microsomeswere prepared by the method of van der Hoevenand Coon'0 from the livers of 3 month old male NewZealand White rabbits and stored at -70°C. Fornon-induced microsomes, the animals had access adlibitum to plain tap water for drinking, but forphenobarbital induction this was replaced by tapwater to which sodium phenobarbital (0.1% w/v)was added. Induction by 83-naphthoflavone was

achieved by intraperitoneal injections daily for threedays at 20 mg/kg as a 1% (w/v) suspension in corn

oil.All chemicals were of AnalaR grade or next high-

est available purity, and were obtained from BDH,Poole, Dorset, unless noted to the contrary. (Trisbuffer used was "i Trizma" base enzyme grade mater-ial from Sigma (London) Chemical Co, Poole.)Ethoxyresorufin and resorufin were gifts from Dr TC Orton, ICI Pharmaceuticals Division. Benz-phetamine HCl was a gift from Dr G G Gibson,University of Surrey. 8-naphthoflavone was pur-chased from Aldrich Chemical Co, Gillingham.

Salmon, Nash, Walklin, and Freedman

SPECTROPHOTOMETRIC ASSAY OFDEHALOGENATIONStored microsomes were thawed gently and dilutedwith buffer (Tris 50 mM, EDTA 1mM, pH 7-5)which had been deoxygenated by bubbling withnitrogen for one hour. The reaction mixture wasprepared with the following components, also indeoxygenated buffer: microsomes (1 mg/ml),D-glucose 30 mM, Catalase, 2000 units/ml (frombovine liver, purchased from Sigma), glucose oxi-dase 5 units/ml (from Aspergillus niger, type V, alsopurchased from Sigma), NADPH 500 ,umol. Sam-ples of the resulting preparation were placed in sea-led cuvettes under nitrogen. All transfers were car-ried out under nitrogen with flushed syringes, con-tainers being sealed with silicone rubber septa orstoppers. The cuvettes were placed in a BeckmanActa MVI or a Perkin Elmer 356 spectrophotome-ter fitted in both cases with a scattering sampleattachment. Absorbance at 340 nm was monitored,a "'baseline" rate of change without substrate beingestablished first. The substrate was added to thesample cuvette as a solution in 1 IlI spectroscopicgrade ethanol. The change in absorbance at 340 nmwas observed as a function of time, and the initialrate determined. Spectrophotometric assays wereperformed in a similar manner with a reconstitutedcytochrome P-450 system (see below). The kineticparameters Km and Vm., were determined by a com-puter program" which uses the direct linear plot'2and also fits regression lines to the three standardlinear transformations of the Michaelis-MentenEquation. The halocarbon susbstrates wereobtained from Aldrich Chemical Co, Gillingham.To study the effect of SKF525A on the reaction of

hexachloroethane, the compound (a gift from Smith,Kline and French, Welwyn) was added in buffer. Inthe similar experiment with carbon monoxide, thereaction mixture was saturated with the gas bybubbling for two minutes.

IDENTIFICATION OF REACTION PRODUCTSFor aerobic incubations, 25 ml flasks sealed withsilicone septa were prepared, containing the follow-ing reagents in a total volume of 10 ml: KCI (75mmol), D-glucose-6-phosphate (5 mmol), NADP+(250 ,umol), nicotinamide (4-5 mmol), glucose-6-phosphate dehydrogenase (0-25 units/ml), in Trisbuffer (as above). For anaerobic incubations, anoxygen scavenging system was incorporated in thereaction system. This consisted of D-glucose, catal-ase, and glucose oxidase, as used in the spec-trophotometric assay. All solutions used inanaerobic incubations were deoxygenated beforeuse, and the headspace of the flask was purged withnitrogen.



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Dechlorination ofhalocarbons by microsomes and vesicular reconstituted cytochrome P-450 systems 307

'4C-hexachloroethane (final concentration 2 ,uM,41-44 ,uCi radioactivity) was added in 20,l ethanolto the liquid phase and the flasks incubated at 37°Cin a shaking water bath. Flasks were sampledimmediately after injection of substrate and after145 minutes. Control incubations with boiled micro-somes were run concurrently. The headspace wassampled (1 ml) with a gas-tight syringe, and ana-lysed by gas liquid chromatography. Two columnswere used as necessary to separate and identify thelabelled metabolite peaks. These were a 15m x2mm id glass column packed with 5 5% OV101 onSupelcoport (100-200 mesh), maintained at 80°C,and a 2 m x 2 mm id glass column packed with60/80 Carbopack B/1% SP1000 (Supelco Inc),maintained at 175°C. Gas chromatography materi-als were obtained from Phase Separations Ltd(Clwyd). A Pye 104 gas chromatograph with flameionisation detector and a radiochemical detector(ESI Nuclear) was used. Metabolite peaks wereidentified by comparison of retention times and bycochromatography with authentic standards. Thegas-tight syringe was purged with nitrogen andchecked for carry over between samples.The radiolabelled hexachloroethane (21-5 mCi/

mmol) used was obtained from Physics andRadioisotopes Services, ICI Petrochemicals andPlastics Division, Billingham. This material was pur-ified by gas liquid chromatography and had aradiochemical purity of better than 98%. No impur-ity peaks were detected on analysis of the materialby gas liquid chromatography, either with the flameionisation or radioactivity detectors.

RECONSTITUTED SYSTEMSPurified preparations of rabbit cytochromes P-450(LM2 and LM4) were derived by a procedureinvolving polyethylene glycol fractionation of cho-late solubilised, pyrophosphate washed microsomes,followed by ion-exchange chromatography onDEAE-Sephacel, adsorption chromatography onspheroidal hydroxyapatite and calcium phosphate(all in the presence of Renex 690), treatment withAmberlite XAD-2 to remove detergent, andchromatography on Octyl-Sepharose 4B. Themethod is modified from the procedures of van derHoeven and Coon'0 and Wolf et a7: full details aregiven by Walklin.'3 NADPH:cytochrome P-450reductase was purified by the method of French andCoon.'4 Reconstruction of these proteins in vesicleswith dilaurylglycerylphosphatidylcholine (FlukaAG, Buchs, Switzerland, synthetic, 98% pure) wasby the cholate gel filtration method.'5 The vesicleseluted at the void volume from columns of SephadexG50 (Medium) and electron microscopy showedthem to be roughly spherical, having diameters in

the range 150-200 nm. SDS-polyacrylamide gelelectrophoresis analysis showed that close to 100%of the added enzymes had been incorporated intothe vesicles.

OTHER ASSAYSMixed function oxidase activities towards stand-ard substrates were assayed: ethoxyresorufinO-deethylation,'6 and benzphetamine N-demethylation.'7 Protein concentrations were meas-ured by the method of Lowry et al'8 modified for usewith membrane proteins,"9 using crystalline bovineserum albumen as a standard. Cytochrome P-450content was assayed spectrophotometically20: thespecific cytochrome P-450 contents of the purifiedpreparations used in this work were: LM2 12-5nmol/mg, LM4 1 1-7 nmol/mg. NADPH:cytochromeP-450 reductase was assayed using cytochrome c asoxidant2'; the stock preparation used here had anactivity of 52 5 units/mg. (One unit catalyses thereduction of 10 ,uLmol cytochrome c per minute:0 3 M phosphate buffer pH 7-7, 30°C.)

Results and discussion

The anaerobic assay system allowed the reliabledetermination of rates of cytochrome P-450 depen-dent reductive dechlorination reactions catalysed byliver microsomes. In the absence of added substratethe rate ofNADPH oxidation was negligible, and onaddition of substrate it proceeded linearly for atleast two minutes. Using rat liver microsomes withhexachloroethane as substrate, 98% was inhibitedby saturation of the assay mixture with CO and 37%inhibited by 100,umol SKF 525A. When phenobar-bital induced rabbit liver microsomes were used theCO inhibition approached 100%.The incubation of microsomes (1-377 mg/ml, 1-4

nmoles P450 per mg protein) and an NADPHgenerating system with hexachloroethane followedby gas chromatographic analysis of the headspaceshowed the formation of tetrachloroethane as themajor product, under conditions of normal oxygenpartial pressure and also anaerobically (fig 1). Therate under anaerobic conditions seemed to be grea-ter than in the presence of oxygen: although exactquantitation of total product is not possible using theheadspace method, the amount of product formedunder aerobic conditions appeared to be between10% and 20% of that formed under anaerobic con-ditions. A second product, trichloroethylene, wasobserved under aerobic conditions in small quan-tities, but was produced in larger amounts in theanaerobic incubation. The other product observedwas pentachloroethane. Under aerobic conditionsthe peak corresponding to this compound was no



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Tetrachloroethane

LiCX0o RadiodemicalAnaerobic Anaerobict= 2 3 h t=2 3 h Boiled microsomecontrol

Fig 1 Gas liquid chromatography ofheadspace productsofa dechlorination reaction catalysed by rat livermicrosomes under anaerobic conditions. Conditions ofincubation were as described in text, and chromatographywas on a column consisting of5% OV101 on Supelcoport.

larger than that seen in the boiled microsome con-trol incubation, but a significant increase in theamount of this material in the headspace (150%)was seen under anaerobic conditions. The presenceof significant amounts of material corresponding toproducts in the boiled microsome control incuba-tions was noted, but this observation was not repe-ated in control incubations lacking only substrate ormicrosomes. It was therefore concluded that in addi-tion to the enzyme catalysed reaction, a small cataly-tic effect is shown by a heat-stable or heat generatedcomponent of the boiled microsomes, possiblyFe"'. Substantial amounts of radioactivity wereshown to remain in the aqueous phase after thecomplete incubations, some but not all of which was

V 0

20 40 60Pentachloroethane (,mol )

Fig 2 Reductive dechlorination ofpentachloroethanecatalysed by rat liver microsomes. Initial velocities observedin spectrophotometric assay described above are displayedas a function ofsubstrate concentration.

unchanged hexachloroethane -"4C.The inhibitor effects and the nature of the pro-

ducts indicate that the reaction being assayed spec-trophotometrically is a cytochrome P-450 depen-dent reductive dechlorination. It has been concludedon the basis of similar observations22 that these pro-ducts resulted from a two electron reduction of thesubstrate. The reaction resembles the reductivedechlorination of carbon tetrachloride except thatthe intermediate radical product is readily suscept-

Table 1 Kinetic parameters for reductive dechlorination ofhalocarbons by control rat liver microsomes

Compound Km (,uM) V nmol NADPH/min V nmol NADPH/min(mmg'fprotein) nmle P-450

Carbon tetrachloride 9-6 3.35 2-15Hexachloroethane 6-0 3-55* 2-41Pentachloroethane 25-1* 4-35* 3*351,1,1,2-tetrachloroethane 20-4* 3-41 2-621,1,2,2-tetrachloroethane a) 'a) (a)1,1,1-trichloroethane b) (b) b)1,1-dichloroethane a) a) a)1,2-dichloroethane a) (a) la)1,1, 1-trichloro-2,2,2-trifluorethane 11-7 1-78 1-371,1,1,2-tetrachloro-2,2-difluoroethane 25 9 4-49 3.45

Kinetic parameters were derived from Direct Linear, Eadie-Hofstee, and Lineweaver-Burk plots of the data. The Lineweaver-Burk plot isknown to be most prone to error when there is experimental scatter in data. Parameters quoted are those obtained from a regression line ofbest fit to data plotted as an Eadie-Hofstee plot; except in a few cases (*) the values obtained from Direct Linear plot are within 10% ofvalues quoted.(a) Rates of NADPH oxidation observed were too low to allow determination of kinetic parameters.(b) NADPH oxidation was observed at a low rate, but protein precipitation interfered with measurements at substrate levels high enoughto allow determination of kinetic parameters.

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Dechlorination ofhalocarbons by microsomes and vesicular reconstituted cytochrome P-450 systems 309

Table 2 Water: octanol partidon data

Compound Log P

Carbon tetrachloride 2-96Hexachloroethane 4-62Pentachloroethane 3-691,1, 1,2-tetrachloroethane 3*051,1,2,2-tetrachloroethane 2-721 ,1,1-trichloroethane 2 501,1-dichloroethane 1-801,2-dichloroethane 1-481,1,1-trichloro-2,2,2-trifluorethane 3-26

Log P is a calculated octanol:water partition coefficient, derived byMr P J Taylor, ICI Pharmaceuticals Division, using the method ofLeo et al.23

ible to further reduction to the olefin in the case ofthe two-carbon substrates, whereas for carbon tet-rachloride .this material is not so readily subject tofurther metabolism and tends to accumulate. Forboth the haloethanes and carbon tetrachloride thisreductive reaction, although more readily detectedunder anaerobic conditions, continues at a substan-tial rate even at the atmospheric partial pressure ofoxygen, which is higher than that to be expected insome tissues-for example, those parts of the liveracinus closer to the central vein. It is therefore sup-posed that this type of reaction is of physiologicaland pathological importance.The rate of reductive dechlorination by control rat

liver microsomes was assayed spectrophotometri-cally with a large number of haloethane substrates,at concentrations between 2 and 500 ,umol. In gen-eral the data are in good agreement with theMichaelis-Menten equation (see fig 2). These datawere analysed to provide values of Km and Vma,

3/2 /

0.2 0.4 06 0.8 10(1,13 -TricNoroethane) mmol _

Fig 3 Reductive dechlorination of 1,1,1-trichloroethanecatalysed by purified rabbit liver cytochrome P-450 (LM2).Conditions for the spectrophotometric assay were asdescribed for vesicular reconstituted systems, initialvelocities being presented as a function ofsubstrateconcentration.

Table 3 Kinetc parameters for reductve dechlorination ofchloroethanes by a vesicular reconstituted system

Compound Km (pM) V nmol NADPH/minnmle P4SO

Hexachloroethane 50 2-39Pentachloroethane 104* 1'481,1,2,2-tetrachloroethane 119 1i661,1,1-trichloroethane 360 4-331,1-dichloroethane 802 1-27

See legend to table 1.

which are reported for a number of substrates intable 1. Similar observations were made for rabbitliver microsomes, although a rather higher absoluterate was observed, especially after phenobarbitalinduction.

With both rat and rabbit microsomes the majorfinding is that there is limited variation in Vm., butconsiderably greater variation in K.. The lack ofdependence of Vm. on structural features of thehaloethanes suggests that in this reductive dechlori-nation the substrate reaction is not rate limiting. Theinterpretation of K. values for the interaction ofnon-polar substrates with membrane boundenzymes is complicated by the partition of the subs-trate between the aqueous and the membranephases; the values obtained neglect this partitionand are therefore not true measures of the dissocia-tion constant describing the equilibrium betweenfree substrate and enzyme/substrate complex.Hence it is dangerous to attempt structure/activityinterpretations of these crude Km values. In generalit is found that the Km values decrease with increas-ing extent of chlorination of the substrates; this mayreflect the increasingly non-polar nature of the morehighly substituted substrates, and hence their grea-ter partition into the membrane phase and theirconcentration in the environment of the enzyme.The polarities of the various compounds are illus-trated in terms of their water-octanol partitioncoefficients (measured or derived) given in table 2.If it is assumed that substrates partition betweenbulk aqueous phase and membrane as determinedby these constants and that substrate in the mem-brane phase then equilibrates with the substratebinding site on the cytochrome, the "true" Kmdescribing this binding equilibrium is given by:

KmaPP *p x

1 +Pxwhere Kapp is the value derived directly from kine-tic studies, P is the partition conefficient and X is thevolume fraction occupied by the membranephase.2425 Using this approach the true Km values



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for the chloroethanes with rat liver microsomes arein the region of 16 mmol.

Involvement of particular cytochrome P-450species in the reductive dechlorination is suggestedby studies with liver microsomes from animals pre-treated with inducers. Microsomes from,8-naphthoflavone induced rabbits did not catalysethe reaction at a measurable rate with any of the sixhaloethanes tested, and microsomes from controlrabbits gave a measurable rate of reaction at highconcentrations of hexachloroehtane only. On theother hand, those from phenobarbital induced rab-bits catalysed the reaction with several compounds.Km values for these microsomes ranged from<5 umol for hexachloroethane to about 80 ,molfor 1,1,2,2-tetrachloroethane.These findings suggest that the reductive

dechlorination reaction is preferentially catalysed bya cytochrome P-450 isoenzyme that is induced byphenobarbital, possibly the major phenobarbitalinducible form. This hypothesis was tested by theisolation from rabbit liver microsomes of the majorphenobarbital induced isoenzyme (LM2), and theisolation of the major aromatic hydrocarbon induc-ible form (LM4) for comparison. These proteinswere isolated rapidly and in good yield by a newcombination of chromatographic techniquesdescribed fully elsewhere.'2 The purified cyto-chrome P-450s were reconstituted with and withoutrabbit liver NADPH:cytochrome P-450 reductase,in defined lipid bilayer vesicles of dilauryl phos-phatidyl choline. Vesicles containing LM2 andreductase were active in the NADPH dependentdemethylation of benzphetamine, but not theNADPH dependent de-ethylation of ethoxy-resorufin, whereas the opposite was true of vesiclescontaining LM4 and reductase. No mixed functionoxidase activities were observed with vesicles con-taining cytochrome P-450 alone or reductase alone.

Vesicles containing cytochrome P-450 LM2 andreductase were active in the NADPH dependentreductive dechlorination or haloethanes. Figure 3shows kinetic data obtained in the reaction with1,1,1-trichloroethane: kinetic data for a range ofsubstrates are shown in table 3. Interestingly, vesi-cles containing LM2 and reductase catalyse reac-tions of a slightly wider range of haloethane subs-trates at measurable rates than do phenobarbitalinduced microsomes (table 3 cf table 1). Compari-son of the kinetic data with various substrates showsthat again there is little variation in Vm,, (3-fold),and considerably greater variation in the Km (16-fold). As is the case with microsomes, Km valuesdecreased with increasing extent of halogen sub-stitution. Adopting the approach described above tocalculate "true" Km values yields for all substrates


values in the region of 80 mM (+50%). Figure 4shows the effect of log P in compensating for thevariation in apparent K., assuming the substratesbind from the membrane phase. Thus in this systemit appears that neither Vm,, nor the "true" Km isstrongly dependent on the nature of the substrateand that the rate determining step may be indepen-dent of substrate. No activity was observed with ves-icles containing LM2 alone or reductase alone, norwith vesicles containing LM4 and reductase. Thereaction is therefore catalysed only by certain cyto-chrome P-450 isoenzymes, and is entirely depen-dent on a flow of electrons from NADPH viaNADPH:cytochrome P-450 reductase. The possibil-ity that NADPH, cytochrome b, and NADPH:cytochrome b5 reductase could also participate aselectron donors to cytochrome P-450 was not inves-tigated.The data presented here are the first direct evi-

dence that the reductive dechlorination pathwayshown for a wide range of haloethanes is a specificproperty of certain cytochrome P-450 isoenzymes.

800- M* KMEM(50 -130 mmol)500- ~~~~AG0 KM ( 2-800FmoI)

* K APP(50-800,umol)

100

10- * *\50- A

5. A A

0

° t2 t t 3t 4 t 5CH3CHCI2 CHCI2CHCI2 CC3CC13

CH3CCI3 CHCI2CCI3

Fig 4 Variation ofKm with log P when calculated on basisoftotal substrate (-), substrate present in the membranephase (A), and substrate present in the aqueous phase (0),partitioning ofsubstrate being calculated as described indiscussion.



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Dechlorination of halocarbons by microsomes and vesicular reconstituted cytochrome P-450 systems 311

The Science and Engineering Research Council andImperial Chemical Industries plc (Central Toxi-cology Laboratory: director, Dr I F H Purchase)have supported this work by providing a studentshipunder the CASE scheme for C M Walklin. Theinvestigations were carried out partly at the Biologi-cal Laboratory, University of Kent, and partly at ICICentral Toxicology Laboratory. Our thanks are dueto Dr C Elcombe (ICI) for making various experi-mental facilities available and for helpful discus-sions. We also thank Dr P R McIntosh and Dr C RWoy for advice on methods of purification of cyto-chrome P-450, and Mr P J Taylor (ICI Pharmaceut-icals Division) who calculated the log P values forus.

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Dechlorination ofhalocarbons by microsomes and vesicular