(CANCER RESEARCH 39. 3942-3947. October 1979]0008-54 72 / 79 /0039-OOOOS02.00

Formation of Carbonyl Chloride in Carbon Tetrachloride Metabolism byRat Liver in Vitro1

Harshida Shah, Sally P. Hartman, and Sidney Weinhouse2

Fe/s Research Institute. Temple University School of Medicine. Philadelphia, Pennsylvania 19140

ABSTRACT

In order to identify intermediates of CCI4 metabolism, whole,suitably fortified rat liver homogenates were incubated with14CCl4 in the presence and absence of "pools" of unlabeled

suspected intermediates. In the presence of NADH or NADPH,incorporation of radioactivity was rapid and substantial in CO2,lipid, protein, and the acid-soluble fraction. It was not influ

enced by the presence of large pools of unlabeled chloroformor formate, thus excluding these substances as obligatoryintermediates. However, when incubated with L-cysteine, radioactivity incorporation in the acid-soluble fraction was almostdoubled, and about one-third of the radioactivity of this fractionwas identified as 2-oxothiazolidine 4-carboxylic acid. This sub

stance is formed chemically by condensation of cysteine withcarbonyl chloride and has been identified previously by othersas a product of chloroform metabolism by liver microsomes inthe presence of L-cysteine. Based on current knowledge of

CCI4 metabolism, the following aerobic pathway is envisioned:microsomal cleavage to Cl~ and -CCI3 and oxidation of the

latter to the unstable intermediate, CI3COH, which loses HCI toyield COCI2. COCI2 is likely to be the major source of CO2 fromCCU but is probably not the intermediate that binds to lipid andprotein. The addition of glutathione had no effect on CCI4metabolism in rat liver homogenate, suggesting that glutathioneS-transferases, which catalyze other dehalogenation reactions,

do not play a role in CCI* metabolism.

INTRODUCTION

Carbon tetrachloride is a potent hepatotoxin and hepatocar-

cinogen in several species including humans (9) and is aprototype of a large number of toxic organohalides the massiveindustrial production and widespread use of which make themenvironmental hazards of great public concern. Despite awealth of data on its toxic effects, there is as yet little definitiveinformation on its metabolism or its mode of carcinogenicaction (6, 23). Like many carcinogens, it is activated for he-patotoxicity by the mixed-function oxidase system (6, 23);

however, unlike many other carcinogens, it binds minimally toDNA (5, 24, 30, 35) and thus far has not been found to bemutagenic in microbial test systems (4, 16). Among reportedmetabolic reactions in liver are conversion to CO2 (18, 26),reduction to chloroform (2, 5, 7, 29) and hexachloroethane (5,29), and binding to lipids and protein (7, 8, 24, 29, 30, 33). Inthe presence of oxygen, it enhances lipid peroxidation andmalonaldehyde formation and destroys the endoplasmic retic-

1This work was aided by Grants BC-74 from the American Cancer Society

and CA-10916 and CA-12227 from the National Cancer Institute, Department ofHealth. Education and Welfare.

2 To whom requests for reprints should be addressed.

Received March 21. 1979; accepted June 25. 1979.

ulum, together with its associated enzymes (23). The trichlo-romethyl free radical, -CCI3, has been proposed as the keycausal agent for these toxic effects (23).

The present study was undertaken to shed light on themetabolism of carbon tetrachloride, with the hope of furtheringour understanding of its carcinogenicity. We have used thewhole, unfractionated rat liver homogenate as a model experimental system to define the conditions for its conversion toCO2, binding to lipid and protein, conversion to water-soluble

intermediates and to identify possible metabolic intermediates.

MATERIALS AND METHODS

Preparation of Liver Homogenate. Male Sprague-Dawley

rats, weighing 200 to 250 g, obtained from the Charles RiverBreeding Laboratories, Inc., Wilmington, Mass., were decapitated; the livers, weighing approximately 12 g, were homogenized in 2 volumes of 0.25 M mannitol by 4 or 5 passes of aTeflon pestle in a coaxial, motor-driven homogenizer, while

cooling in ice.Carbon Tetrachloride. The 14CCI4was obtained from Amer-

sham/Searle Corp., Arlington Heights, III., and for most experiments was diluted with unlabeled reagent grade CCI4 to giveapproximately 60,000 dpm//imol of 14CCI4.For the high-activ

ity experiments used for identification of 2-oxothiazolidine 4-carboxylic acid, the 14CCI4 was 10 times more active at600,000 dpm//imol. The 14CCI4 was uniformly added as a

solution in 0.2 ml dimethyl sulfoxide (DMSO) to facilitate accurate measurement and to enhance its solubility in the medium. At this concentration of DMSO, there were no deleteriouseffects on O2 uptake or on 14CCI4 metabolism. The 14CCI4

solutions were assayed for purity of CCI4 by gas chromatog-

raphy.Nicotinamide adenine nucleotides were obtained from Sigma

Chemical Co., St. Louis, Mo. (best available grade), and otherreagents were top grades obtained from usual commercialsources. Glass-distilled water was used for all reagents.

The following basic procedure for incubation and subsequentseparation of products was subject to minor modifications asdictated by individual experiments. A volume of 0.5 ml of thehomogenate, equivalent to 167 mg of liver, was added to ice-chilled 18-ml Warburg vessels carrying a center well and asingle glass-stoppered side arm. Components were added tothe main compartment to make a final volume of 2.5 ml of amixture containing 24 HIM KCI, 40 mM potassium phosphatebuffer (pH 8.2), 2 mM MgCI2, 2 mM ATP, 1.6 mM NADPH, and10 /imol 14CCI4.A fluted filter paper strip (18 x 40 mm) was

placed in the center well, 0.2 ml of 5 M NaOH was added toabsorb CO2,3 0.2 ml of 2 M H2SO4 was pipetted into the side

3 We avoided the use of organic bases such as Hyamine to absorb CO2because they absorbed <4CCI< vapor as well and thereby complicated the

experiments.

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Carbon Tetrachloride Metabolism in Liver

arm, 0.2 ml of the 14CCU in DMSO was added last, and the

flasks were quickly placed on manometers and immersed in abath at 37.5°. After temperature equilibration for 5 min, the

taps were closed, and the flasks were shaken at approximately100 oscillations/min. Readings of O2 uptake were taken atregular intervals for 30 min;4 then acid was tipped in and the

shaking was continued for 15 min to allow complete absorptionof metabolic CO2. Two ml of toluene were added through theside arm to collect unreacted 14CCI4; the flasks were quickly

removed, covered with foil, and chilled in ice. The filter paperswere removed, the vessel contents were transferred to 15-mlcentrifuge tubes and spun briefly, and the toluene layer wasdrawn off. Portions were taken for radioactivity assay in atoluene:POPOP:PPO scintillation mixture; in some instances,1.0 ftl was injected into a Glowall gas Chromatograph (GlowallCorp., Willow Grove, Pa.) with injection port at 37°,an electroncapture detector at 70°,and voltage at 35 V. We found in thismanner that approximately 60 to 70% of the added 14CCI4was

recovered unchanged with a retention time of 1.3 min, andthere was no detectable chloroform or hexachloroethane in therecovered 14CCI4.

The alkali-soaked papers were dried overnight in a vacuum

and extracted with 1.8 ml water, and 1 ml was counted in 10ml of scintillation mixture with the following composition: p-xylene, 2250 ml; Triton X114, 750 ml; PPO, 9 g; and POPOP,0.6 g.

The aqueous layer was extracted with two 1-ml portions ofethyl ether to remove residual traces of 14CCL,,and the aqueous

layer (together with a thin layer of semisolid that collected atthe interface) was separated from the tissue residue by cen-trifugation. A measured portion was counted in Formula 963scintillator fluid.

The tissue residue was extracted successively 3 times withethanol and ethyl ether, the extracts were evaporated to dry-ness in a stream of N2 and taken up in chloroform:methanol (2:1), a portion was evaporated to dryness as above in a scintillation vial, and this lipid fraction was counted in a toluenescintillator.

The residue was dried thoroughly, and a weighed portionwas dissolved in Protosol and counted in Bray's solution. This

fraction was termed the protein fraction, since others reported(5, 24, 30, 35), and we have confirmed, that there is nosignificant activity in the nucleic acid fraction.

All of the radioactivity assays were made with an Intertechnique scintillation counter and were corrected for efficiency byuse of external standards.

Determination of Radioactivity Present as Formate. Metabolic formate was recovered from the acid-soluble fraction bythe highly specific oxidation to CO2 by HgSO4 (34), absorbingthe liberated CO,, in alkali and counting as described above.To facilitate recovery of the small amounts of formate present,0.1 mmol of nonlabeled formate was added as carrier. Yieldsof CO2, determined by precipitation and weighing as BaCO3,ranged from 73 to 112%.

Synthesis of 2-Oxothiazolidine 4-Carboxylic Acid. The syn

thesis was conducted essentially as described by Kaneko efal. (12), except for isolation of the product by repeated extraction with ethyl acetate from the acidified solution rather than byconcentration of the aqueous reaction mixture. The producthad a melting point of 173-174° (reported by Kaneko ef a/.,171 -172.5°). Analysis: C, 30.37; H, 3.03; S, 20.08; calculated

for C4H5NSO3: C, 32.65; H, 3.40; S, 21.76; neutralizationequivalent, 154; calculated, 147.

Identification of 2-Oxothiazolidine 4-Carboxylic Acid as a

CCU Metabolite. Two experiments were conducted with 10vessels, each containing 10 /unol 14CCI4and 12.5 /tmol (5 HIM)

L-cysteine, incubated and treated as described in Table 1,except for a 10-fold higher radioactivity; i.e., 6 x 106 dpm/vessel. The acid-soluble fractions from 4 vessels were com

bined to give a total of 59,100 dpm. This solution was adjustedto pH 1.0, extracted with ethyl acetate, reextracted into theaqueous phase with 0.02 M K2HPO4 buffer (pH 8), acidified,and reextracted in ethyl acetate. The process was repeatedtwice, after which essentially all of the ethyl acetate-solubleradioactivity was drawn into the alkaline aqueous phase andwas reextracted into ethyl acetate after acidification. The ethylacetate-soluble material had 18,900 dpm or 32% of the initialradioactivity. A portion was streaked on a soft thin-layer 10- x20-cm silica chromatography plate, developed with chloroform:ethanohacetic acid (80:20:10), and counted. Ninety-eight % ofthe applied radioactivity appeared in bands with RF between0.45 and 0.65, exactly where the authentic acid migrated, asvisualized by staining with iodine (Chart 1).

Crystallization to Constant Specific Activity. Another ethylacetate extract of the acid-soluble fraction, partially purified asdescribed above, containing 10,900 dpm was evaporated todryness, taken up in ethanol, 98 mg of synthetic acid wereadded as carrier, and the mixture was subjected to 4 successive recrystallizations from ethyl acetate:hexane. The recoveries of each of the 4 crystallizations were: 74.5, 58.7, 46.5,and 28.3 mg, and the corresponding specific radioactivitieswere 101, 113, 115, and 108 dpm/mg. The calculated radioactivity of 98 mg containing a total of 10,900 dpm = 10,900798 = 111 dpm/mg.

20000CL

Q 10000"

* " ~ ~/u.0 0.2 0.4 0.6 0.8 1.0

Rf

4 Although O.. uptake was not an important parameter of CCI4 metabolism, it

was convenient to measure and served as an index of tissue viability. It wasdetermined in all experiments but was omitted from those tables in which nosignificant changes occurred. In all experiments, O.. uptake was linear throughoutthe entire 30-min incubation.

Chart 1. Chromatography of acid-soluble "CCU metabolite, purified by re

peated acid:base extraction as described in the text, on a 10- x 20-cm silicathin-layer plate, using chloroform:ethanol:acetic acid as the developing fluid. Thehorizontal line above the peak indicates the range of yellow color developed withiodine vapor when the authentic material was chromatographed in parallel withthe metabolic material. For radioactivity counting. 1-cm bands were scraped, andthe product was eluted in scintillation vials with 1 ml ethanol and xylene scintillation fluid added.

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H. Shah et al.

Mass Spectrometry. Another portion of the acid-solublefractions was purified as above by acid:base extraction andthin-layer chromatography followed by repeated acid-base ex

traction, and portions were used for mass spectrometry on aHitachi-Perkin Elmer RMU-6H instrument with direct probeinlet. Source temperature was 200° and best probe temperature was 100 to 200°. lonization voltage was 70 eV, acceler

ation voltage was 1.8 kV, and current was 70 /ia. As shown inChart 2, the synthetic material had major peaks at 147 (themolecular ion), 102, and 74. Despite some extraneous peaks,the presence of peaks at 147, 102, and 74, with nearly thesame peak height ratios as given by the synthetic compound,provides further evidence for the metabolic formation of 2-oxothiazolidine 4-carboxylic acid from CCU. This same productgave a single radioactive peak and a single iodine vapor spotat RF 0.58 on thin-layer chromatography with a GF hard-layer

silica plate, developed as described earlier.

RESULTS

Properties of the experimental system are depicted in Table1. Incubation at 37.5°with 10 ¿imolof CCU and 1.6 (TIMNADPH

resulted in rapid CCU metabolism, with substantial conversionto CO2 and water-soluble products and binding to lipids and

Table 1Eltect ol NADH and NADPH on ' ' CCI, metabolism in rat liver

Experiments were conducted as described in the text with added nucleotideas indicated, and incubations were conducted for 30 min at 37.5°. Each vessel

contained 2.5 ml fluid consisting of the basic medium, 0.5 ml of liver homogenateequivalent to 0.167 g tissue, 10 nmul of 14CCI4(approximately 600.000 cpm),

and NADH, NADPH. or both, each at 1.6 rtiM In this and subsequent tables,values for O.. uptake are given in nmoi per g liver and other values are in nmolCCI»carbon per g liver ±S.E. of 3 separate experiments, each run in duplicate.

In Experiment 2. the regenerating system consisted of 5 mw glucose 6-phosphate and 20 units glucose-6-phosphate dehydrogenase.

NucleotideaddedExperiment

1NoneNADHNADPHNADH

+NADPHExperiment

2NADPHNADPH

+regeneratingsystemO2

uptake446060607663±

5±2±3±2±

3±2CO?27373464472572460±

5±17±33±21±

13±2Acid-solu

ble37159210215232235±±±±±¿610e11818Bound

toLipid14

±59±72±65±87

±58±242274Protein7779286111107±

2±4±4±3±2±

6

protein. By 30 min, approximately 7.5% of the added 14CCU

was accounted for in these products; 60 to 70% of the initialradioactivity added was recovered by extraction of the flaskcontents with toluene. That this was unreacted '"CCI* was

established by gas chromatography, which yielded a singlepeak with a retention time of 1.3 min. O2 uptake was measuredas a criterion of tissue viability. It was strictly linear for 30 minand about half-maximal without added nucleotides. However,metabolism of 14CCI4to CO2, and to acid-soluble material, and

protein and lipid binding with no added nucleotide were lessthan 10% of that at the optimal concentration of 1.6 mM. Noremarkable differences were observed between NADH andNADPH at 1.6 mM, but the latter was the better substrate atlower concentrations. There was no significant additive effectwhen both nucleotides, each at 1.6 mM, were present, nor didthe addition of an NADPH-regenerating system consisting ofglucose 6-phosphate and glucose-6-phosphate dehydrogenase enhance '"CCU metabolism.5

Formate Not an Obligatory Intermediate. To assess the roleof formate as an intermediate of CCU metabolism in liver, theclassical isotope trapping technique was used. Experimentswere conducted in which '"CCU was incubated with nonlabeled

formate, on the assumption that any metabolic formate wouldbe "trapped" by the pool of unlabeled formate and thereby

lower the radioactivity in the metabolic products and enhanceradioactivity in the acid-soluble fraction. The data of Table 2

are typical of many experiments which failed to show sucheffects. Whether 1 or 10 jumol 14CCUwere added, the addition

of 1 or 10 /imol of formate did not lower the incorporation ofradioactivity in any of the products and did not increase theradioactivity in the acid-soluble fraction These results are

considered to exclude formate as an obligatory intermediate.Formation of Chloroform. Under the conditions used for

'"CCU metabolism, analysis of the toluene fractions by gas:

liquid chromatography revealed little or no chloroform production. However, since chloroform is metabolized more rapidlythan 14CCU in rat liver (26), it could have been formed andutilized. "Trapping" with nonlabeled chloroform was therefore

used to make a more definitive assessment of its formation.

Table 2Effect of added formate on ' ' CCI, metabol'sm in rat liver

Experiments were conducted as described in Table 1, with either 1 or 10 firnol"CCU and either 1 or 10 firnol sodium formate. Values given are averages ±

S.E. of 5 separate experiments. O2 uptakes were not affected and are omitted.

Chart 2. Mass spectra of authentic 2-oxothiazolidine 4-carboxylic acid (top)and the "CCU metabolite (oofrom) Mass spectra were obtained as described in

the text. Top, results with 4.7 pg of the synthetic acid dissolved in 10 n\ of ethylacetate and evaporated in the mass spectrometer; bottom, mass spectrumobtained by eluting the radioactive material from a silica plate with radioactivityrepresenting approximately 4 fig of acid. The peak at 149 is an impurity,appearing in blank runs.

BoundtoAdditions1

fimol"CCUFormateNone1

nmol10fimol10fimol

"CCU

FormateNone1

0 /imolCO2125

±24137±26139±27348

±7376±23Acid-soluble42

±744±746±8154

±5145±8Lipid29

±1347±1743±1757

±659±21Protein43

±1155±1147±1383

±670± 8

5 In other experiments not shown, we found that "CCU metabolism was

proportional to its concentration up to 4 mu but was toxic at 10 mM. Neitherphénobarbital nor 3-methylcholanthrene pretreatment influenced "CCU metab

olism. Addition of ^-diethylaminoethyl-2.2-diphenylpentanoate (SKF-525A) hadno effect up to 0.1 mM, but KCN at 1 mM inhibited all metabolic conversions by40 to 50%.

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Carbon Tetrachloride Metabolism in Liver

Experiments were conducted with either 1 or 10 /nmol of 14CCU,

to which were added 0, 1, or 10 /imol of nonlabeled CHCI3. Asshown in Table 3, there was no decrease of radioactivityincorporation into CO2, lipid, protein, or acid-soluble fractions.We assume, therefore, that under aerobic conditions chloroform is formed in minimal amounts, if at all, from 14CCU by rat

liver; or if formed, it does not mix with an exogenous pool ofchloroform.

Formation of Carbonyl Chloride. Recent studies demonstrated that carbonyl chloride (phosgene) is a metabolite ofchloroform in rat liver microsomes. Incubation of CHCI3 in thepresence of L-cysteine led to the formation of 2-oxothiazolidine4-carboxylic acid, a product formed chemically by condensation of L-cysteine with carbonyl chloride (15, 20). To test for

the possible formation of carbonyl chloride in hepatic carbontetrachloride metabolism, we added 5 mM L-cysteine to ourbasic system, together with 10 /imol of 14CCI4. As shown in

Table 4, the presence of cysteine resulted in moderate decreases in CO2 formation and protein binding and somewhatincreased lipid binding, but the most striking effect was apronounced increase of radioactivity in the acid-soluble fraction.

The presence in this fraction of the above-mentioned thioacid was established by the identity of migration of radioactivitywith the synthetic material in thin-layer chromatography (Chart1), by retention of a constant specific radioactivity on repeatedrecrystallization, and by the appearance of characteristic masspeaks when the mass spectrographs of the synthetic andmetabolic material were compared (Chart 2). The mass spec-

Table3Effectof addedchloroformon "CCU metabolismin rat liver

Experiments were conducted as described in Table 1. with either 1 or 10 /imol"CCU and 1 or 10 fimol CHCI3. Values are the averages of 3 separate experi

ments. O? uptakes were not affected and are omitted.

BoundtoAdditions1

/imol '"CCI.

ChloroformNone1mol10

molC02187

±205±184

±8"43Acid-soluble52

±1657±1147±10Lipid64

±1492±2752± 7Protein68

±297±880±20

ChloroformNone10 mol476

±62484 ±29161

±17158 ±1475

±1771 ± 9150

± 7160 ± 2

Mean ±S.E.

Table 4Effect of cysteine and glutathione on "CCI, metabolism in rat liver

Experiments were conducted as described in Table 1, with either 5 mw L-cysteine or 5 mM glutathione and 10 /jmol MCCU. Each value is the average of 3

separate experiments with cysteine and 2 experiments with glutathione. O2uptakes were not affected and are omitted.

AdditionsExperiment

1NoneCysteineExperiment

2NoneGlutathioneCO2580

±48"510

±66426.

648504,690Acid-solu

ble248

±19578±76218,

222210,206BoundLipid145

±13229±3269,

14554,105toProtein155

±1390±12106,

16337,96

Mean ±S.E.

trum exhibits impurities still present in the metabolic product;however, the presence of the molecular ion peak at 147 andthe other major peaks at 102 (the oxothiazolidine ring) and 74(the fragment, —SCH2CHNH) helps to confirm the identity of

the metabolic product, and further support is provided from thesimilarity in the ratios of the peak heights, 36:100:60 versus31:100:80.

Lack of Effect of Glutathione. In contrast with the markedaction of L-cysteine on the incorporation of radioactivity in theacid-soluble fraction, glutathione had no significant effect onthe disposition of 14CCU in these experiments as shown in

Table 4, Experiment 2. We were led to test this substance for2 reasons. Rubinstein and Kanics (26) found that, althoughglutathione had little or no effect on CCU metabolism, it markedly increased the oxidation of chloroform by rat liver homog-

enate; it is becoming increasingly recognized that one or moreof the isozymes of glutathione S-alkyltransferase dehalogenatecertain organohalides (10,11 ). Our results indicate that neitherglutathione nor the S-transferases play a role in those metabolic

conversions measured by us.

DISCUSSION

The formation of 2-oxothiazolidine 4-carboxylic acid in our

system demonstrates the metabolic formation of carbonyl chloride from CCI4 as shown previously for CHCI3 (15, 20) andemphasizes basic similarities in the metabolism of both chlo-

roalkanes. An important question raised by this finding concerns a possible role of phosgene in hepatocarcinogenesis. Itis conceivable that products of lipoperoxidation are responsiblefor CCU hepatocarcinogenesis; indeed, malonaldehyde hasbeen reported to be carcinogenic (27) as well as mutagenic(17). Phosgene, an intermediate of both CHCI3 and CCU metabolism, deserves special consideration as a carcinogen. Asearch of the literature has not yet revealed any data on COCI2carcinogenicity or mutagenicity, although it is highly toxic tolungs exposed acutely or chronically to low levels (19). Its 2highly reactive chlorines suggest that it could act on DMA orother macromolecules in ways similar to those of the bifunc-

tional alkylating agents. The lack of binding of CCU carbon toDMA argues against this possibility, however. Studies on thepossible mutagenicity of phosgene are now under way (conducted by Nobuto Yamamoto of Fels Research Institute).

Mechanism of Phosgene Formation. The formation of phosgene as an intermediate may be placed within the frameworkof our current knowledge of 14CCU metabolism as outlined in

Chart 3. A large body of experimental data indicates that thefirst step is a rapid reductive formation of the trichloromethyl(•CCI3)radical by complexing with one or more of the P-450

cytochromes (21, 23). Anaerobically, this radical may undergoseveral reactions, namely, addition of a proton and electron toyield chloroform (2, 5, 7, 29), binding to lipids (8, 24, 30, 32)and proteins (29, 30) (but not to nucleic acids) (24, 30, 31),dimerization to hexachloroethane (5, 29), and further reductivedechlorination to yield carbon monoxide, presumably via thecarbene, CCI2, as suggested by Wolf et al. (35). Chloroform (1,30) and méthylènedichloride (13, 14, 22, 25) also yield CO,presumably via the same carbene intermediate.

The repeatedly observed formation of chloroform from CCU,both in vivo and in vitro and in several species, might at first

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H. Shah et al.

quite different from that of 14CCU. Moreover, C36CUradioactiv

ity was also stably incorporated into liver lipid and protein,pointing to the CI3C- radical rather than phosgene as thereactive form. Cessi étal.(3) also reported that [14C]phosgene

given to rats labeled the liver proteins. The significance ofphosgene as the form in which CCU carbon binds to lipid andprotein obviously needs further investigation.

Fe2+CCI2+Cr ACKNOWLEDGMENTS

[Fe --CCI3-02~ —- Lipoperoxidation

ConjugationI+2H Molonaldehyde

[Fe3+-CI3COH] +OH"

CO

Acceptor •«Acceptor-MCI

•2HCI +CO-

Chart 3. Pathways of CCU metabolism. Products identified as CCU metabolites are underlined. The electrons utilized in the reactions are assumed to comefrom NADH or NADPH via the flavoprotein cytochrome reductases. Fe2* andFe3* in the above denote the respective ferro- and ferricytochromes.

glance appear contradictory with our results, which indicatethat CHCI3 does not dilute the incorporation of '"CCU carbon

into metabolites. However, CCI4 yields CHCI3 most readily invitro under anaerobic conditions, and its formation is inhibitedby oxygen (7, 29). These findings would suggest minimalformation under normal physiological conditions. It is conceivable that chloroform may have been formed and undergonerapid further reaction, since in a preparation similar to oursRubinstein and Kanics (26) found it to be more rapidly converted to CO? than CCU. However, if CHCI3 is formed as atransient, obligatory intermediate of CCU metabolism, it evidently is metabolized further without mixing with a large poolof free chloroform. Our results would also indicate that CHCI3does not compete successfully for a rate-limiting step in CCU

metabolism, such as the initial binding of the halocarbon tocytochrome P-450 (23, 28). Wolf ef al. (35) found that bindingof CHCI3 to reduced cytochrome P-450 was extremely slow

compared to that of CCU.Aerobic Metabolism of CCU. Aerobically, the -CCI, free

radical could be oxygenated by the microsomal mixed-function

oxidase system to yield trichloromethanol, CI3COH. This substance was suggested by Mansuy ef al. (15) and Pohl ef al.(20) as the precursor of phosgene formed metabolically fromCHCI3; Pohl ef al. (20) clearly demonstrated that the phosgenemust have been formed by an oxygenase reaction. CO2 wouldbe formed by the hydrolytic dechlorination of COCI2, and itseems highly likely that this is the pathway by which themicrosomal oxygenase system converts CCU to CO2.

With the evidence that phosgene is the precursor of CO2from CCU, it is necessary to consider that it might also be oneof the reactive species that bind to lipids and protein. Reynolds(24) showed that 14COCI2 given to intact rats labeled liver

protein (and lipids to a smaller extent), but the pattern was

We are grateful to Bruce Huang of the Smith Kline Corp. and Gordon Hansonof the Department of Chemistry. Temple University, for assistance in the massspectra determinations.

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1979;39:3942-3947. Cancer Res   Harshida Shah, Sally P. Hartman and Sidney Weinhouse 

in VitroMetabolism by Rat Liver Formation of Carbonyl Chloride in Carbon Tetrachloride

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