Lipid Metabolites of Carbon Tetrachloride

ENOGHGoRDIS

From The Rockefeller University, NewYork 10021

A B S T R A C T 5 min after intravenous injection intorats of "C- or 'Cl-carbon tetrachloride, liver lipidswere found labeled. Most of the radioactivity was foundin the phospholipid fraction. The metabolites were shownto comprise a heterogeneous group of branched long-chain chlorinated fatty acids, probably containing thetrichloromethyl side chain. Surviving liver slices alsoformed these metabolites. In a simple chemical systemwhich generates trichloromethyl free radicals, carbontetrachloride added to methyl oleate to form esterswhich behaved like the metabolites during counter-current distribution and urea adduction. The evidencestrongly suggests the formation of these metabolites byfree radical attack on unsaturated lipids. The relation ofthese observations to current theories of carbon tetra-chloride intoxication is discussed.

INTRODUCTION

Poisoning by carbon tetrachloride has often been thoughtto result from its ability as a lipid solvent to destroylipid-rich membranes of cells and subcellular particles.This view is probably incorrect. It fails to explain thevariability in toxic dose, the alteration in susceptibilityto poisoning produced by antioxidants (1, 2), and thegreater toxicity of carbon tetrachloride than chloroform(3). Most striking is Slater's demonstration (4) thatin equimolar doses, fluorotrichloromethane, a liquidsimilar to CCl1 in physical properties and solvent power,is not hepatotoxic at all. Other theories of CCl4 poison-ing which attribute its effects to the unchanged CCl4molecule [e.g., stimulation of catechol release (5), inter-ference with blood flow to the centers of liver lobules(6)] are not supported by recent evidence (7, 8). Muchof this evidence is discussed in Recknagel's comprehen-sive review (9).

Recent efforts have therefore been concentrated on therelation between the metabolism of CCl4 and its toxicity.In 1951, McCollister, Beamer, Atchison, and Spencer(10) showed that while most 14C-CCl4 breathed bymonkeys was exhaled unchanged, a small fraction was

Received for publication 16 July 1968.

converted to radioactive carbon dioxide, chloroform,urea, and one or more unidentified urinary organicanions. This group of products did not in itself suggesta toxic mechanism. In 1961, Butler (11) showed that thein vitro reaction of CCl4 with sulfhydryl compounds ledto replacement of sulfhydryl hydrogen. He inferred thatthe reaction mechanism involved the homolytic cleavageof CC14 into CClM and Cl free radicals with radical at-tack on the susceptible functional groups. In Butler's ex-periments, sulfhydryl groups were oxidized to disulfidewith formation of chloroform. He suggested that in othercircumstances, the trichloromethyl radical itself mightreplace the sulfhydryl hydrogen. Free radical attack onsulfhydryl groups might take place in vivo, but no suchproducts have been identified yet in intoxicated animals.Carbon tetrachloride may poison by means of two otherwell characterized reactions of free radicals: the additionof halomethanes to olefins (12), and the chain per-oxidation of dienes and polyenes (13). In vitro thesereactions are best initiated by heat, ultraviolet light, orcatalytic amounts of organic peroxides. In the caseof addition to olefins, trichloromethyl radicals add to onecarbon of the double bond, and depending on the reac-tion conditions, either Cl or H adds to the other. In thecase of chain peroxidation of dienes and polyenes, aninitiator, which can be the CCMe radical, catalyzes theformation of peroxide radicals, but this reaction is self-perpetuating and proceeds without further catalyst. Inboth cases polymerization can result from one or morecouplings of product radicals. Unsaturated lipids areobvious substrates for both free radical reactions.

Reynolds (14) reported that rat liver lipids werelabeled after feeding either "C- or 'C-carbon tetra-chloride. Microsomal lipids were most heavily labeled.The studies described below demonstrate the labeling ofliver lipids within 5 min after the intravenous injectionof "C- and 'Cl-carbon tetrachloride, confirming theobservations of Reynolds. The metabolites comprise aheterogeneous group of branched chlorinated long-chainfatty acids. Surviving liver slices also formed thesemetabolites. These products share the properties oflipids synthesized by benzoyl peroxide-catalyzed addi-

The 'Journal of Clinical Investigation Volume 48 1969 203

tion of carbon tetrachioride to unsaturated esters. Thestructure of the metabolites strongly suggests theirformation through attack by trichlorornethyl radicals onunsaturated lipids.

METHODSUnlabeled carbon tetrachloride, certified ACS grade, wasobtained from Fisher Scientific Company, Pittsburgh, Pa.,'C-carbon tetrachloride, 7.2 mc/mM, was obtained fromNuclear-Chicago Corporation, Des Plains, Ill., and "6Cl-carbon tetrachloride, 0.35 mc/mM, from the RadiochemicalCenter, Amersham, England. Methyl oleate, 99% pure, andampoules of BF3-methanol, were obtained from AppliedScience Laboratories, Inc., State College, Pa., and Lipomulfat emulsion for intravenous injection from the UpjohnCo., Kalamazoo, Mich. Silicagel G was purchased fromMerck, Darmstadt, Germany, and silicic acid, 100 mesh,A.R., from Mallinckrodt. Liquiflor, a concentrated solutionof 2,5-diphenyloxazole (PPO) and 2-p-phenylenebis (5-phenyloxazole) (POPOP) in toluene, were obtained fromNew England Nuclear Corp., Boston, Mass. A 25-folddilution in toluene provides a solution of PPO 4 g/liter,and POPOP50 mg/liter.

Solvents were redistilled before use, unless otherwisenoted.

Radioactivities of samples containing 14C or 36Cl wereassayed in the double channel Nuclear-Chicago model 720scintillation counter. Discriminator settings were chosen sothat the isotopes could be assayed simultaneously.

Radioactive carbon tetrachloride was administered intra-venously to rats without additional unlabeled carbon tetra-chloride. The tracer was mixed with Lipomul, yielding astable aqueous radioactive preparation, since the tracerdissolved in the fat particles of the emulsion. 14C-carbontetrachloride was supplied in a vacuum ampoule. The topof the ampoule was broken under a layer of Lipomul, per-mitting the radioactive vapor to mix rapidly with theemulsion. 'Cl-carbon tetrachloride, of lower specific activity,was supplied as a liquid, and was added to the Lipomul witha Hamilton microsyringe. To assay the radioactivity of theLipomul-CCl4 mixture, we distributed 5 ,tl in the two phasesystem, heptane (4 ml)-isopropanol (4 ml)-water (3 ml),and counted an aliquot of the upper (heptane) phase.

None of the fat in Lipomul was labeled by mixing withcarbon tetrachloride. An alternative intravenous preparation,CCl4-propylene glycol-water, yielded identical liver metabo-lites, but this system incorporated less tracer and introduceda glycol, so that it was abandoned in favor of Lipomul.

To obtain liver lipids, we homogenized livers or liverslices in chloroform-methanol 2: 1 (v/v). Homogenates werefiltered, the filtrates washed with 0.2 volume of water, andthe lipids recovered by evaporating the chloroform and re-

dissolving the residue. "Phospholipids" refers to lipids pre-cipitated in the cold by adding 9 volumes of acetone to achloroform solution of total lipid extract.

Glyceryl esters were hydrolyzed by refluxing in 4 N HCOfor 3 hr. The reaction mixture was cooled and distributedin about 30 volumes of the two phase system petroleumether-isopropanol-water (4: 4: 3 v/v). Fatty acids wererecovered from the upper phase by evaporating the solventin a rotary evaporator. Methyl esters were prepared fromthe free fatty acids by heating the acids 1 min at 100°C inBF,-methanol. Lipids were not saponified in alkaline alco-hols, since these reagents dehalogenate.

Phospholipids were fractionated by chromatography on

silicic acid according to .the procedure of tIanahan, Dittmer,and Warashina (15). The eluting solvents were evaporatedini a rotary evaporator and the residues dissolved inpetroleum ether before assay of lipid phosphorus (16) and36Cl radioactivity.

Countercurrent distributions of fatty acids and of fattyacid methyl esters were performed using the solvent systemsof Ahrens and Craig (17). For fatty acids, the solventphases were prepared by mixing heptane, 97.5% acetic acid,methanol and acetonitrile (4: 1: 1: 1 v/v) and allowingphase separation. For methyl esters, the phases were ob-tained after equilibration of equal volumes of heptane andacetonitrile. Radioactivities were assayed after counter-current distribution by evaporating an aliquot of each upperphase in a counting vial and redissolving the residue in thescintillation solution.

Branched-chain compounds were distinguished fromstraight-chain compounds by the formation of urea adducts(18, 19). When urea and a straight-chain hydrocarbon aredissolved together in methanol, inclusion complexes pre-cipitate spontaneously. These complexes consist of hollowcylindrical crystals of urea in which the hydrocarbon chainis included. Branching of the chain tends to prevent itsinclusion in the urea complex, and branched compounds re-main mostly in the supernatant. Fatty acid methyl esters weredissolved in hot 15% methanolic urea, and the adductsprecipitated spontaneously overnight at room temperature.The complexed esters and the excluded esters were recoveredseparately by distributing the precipitate and the supernatantin the system petroleum ether-methanol-water.

Thin-layer chromatography on Silicagel G impregnatedwith silver nitrate was used to distinguish saturated fromunsaturated esters. Plates 700 u thick were prepared by amodification of the method of Kaufmann and Wessels (20)

RESULTSIn vivo studiesAs early as 5 min after intravenous injection of radio-

active carbon tetrachloride, rat liver lipids were labeled.The analyses described below were done on lipids ob-tained 15 min after injection. Rats were given either36Cl-carbon tetrachloride or "doubly labeled" carbontetrachloride, a mixture of "C- and 36Cl-CCl4. The useof carbon tetrachloride labeled only with 36Cl permittedsimultaneous comparison of chlorinated metabolites withstandard "C-palmitic acid and its methyl ester duringcountercurrent distribution and urea adduction. Use ofdoubly labeled CC14 permitted calculation of the num-ber of chlorine atoms per molecule of CC14 incorporatedinto lipid.

LIVER LIPIDS AFTER INJECTION OF S'CL-CARBONTETRACHLORIDE

Male rats of the Sprague-Dawley strain weighing 125-150 gm were fasted overnight. They were each injectedintravenously with 1 ml Lipomul containing 8 X 105 cpm3'Cl-carbon tetrachloride. 15 min later the rats weredecapitated, and the liver lipids were extracted. Thelipid extracts contained about 0.5%O of the injected radio-activity. The extracts were pooled, and the phospholipids

204 E. Gordis

precipitated with acetone. 65% of the radioactivity wasfound in the acetone-precipitable lipids. None of theactivity in the acetone precipitate was due to 'Cl-chloride ion, since shaking a wet solution of the phospho-lipids with powdered silver carbonate did not reduce thelipid -radioactivity.

Distribution of radioactivity in phospholipid fractions.To determine if any class of phospholipid was preferen-tially labeled, we fractionated a portion of the acetone-precipitable lipid on a silicic acid column (15). Table Ipresents the elution sequence, and the lipid phosphorusand 'Cl activity of each fraction. From the finding thatall of the phospholipid fractions were labeled, as well asthe labeling of acetone-soluble lipids, it appeared proba-ble that the fatty acids were labeled. The remainder ofthe phospholipid was hydrolyzed, and the fatty acidsrecovered. To this fatty acid mixture, 14C-palmitic acidwas added in an amount chosen to make the "4C and 'Clradioactivities similar. 2/3 of this fatty acid mixturewas then esterified in BF3-methanol.

Countercurrent distribution of labeled fatty acids andmethyl esters. Phospholipid fatty acids containing MCl-acids and added "4C-palmitic acid were distributedthrough twenty transfers in the system heptane-aceto-nitrile-acetic acid-methanol, lower phase 10 ml, upperphase 6.8 ml. 5-ml aliquots of each upper phase wereevaporated in counting vials and the residues dissolvedin scintillation fluid for assay of radioactivity. Fig. 1shows the distribution of the free acids. Fig. 2 shows thedistribution of the methyl esters (heptane-acetonitrile,10 ml upper phase, 10 ml lower phase, 8 ml of upperphases assayed).

The labeled metabolites thus behave as a mixture oflong-chain acids. To show that the chlorinated lipid isnot a single substance, consider the simultaneouscountercurrent distribution of two pure substances, Aand B, with partition coefficients K. and Kb. Call theconcentration of A in the rth tube A., and of B in the rthtube Br. It can be readily shown that if any two adja-cent tubes are compared, say the rt" and the (r + 1)th

TABLE I36CI-Labelling of Liver Phospholipid Fractions after

Administration of 36CI-Carbon Tetrachloride(Silicic Acid Chromatography)

TotalTotal eluted

Fraction eluted lipidNo. Eluting solvent lipid P 36C1

I CHC13-CH30H 7:1 1.2 9II IV 4:1 38.0 26III " 3:2 55.0 48IV " 1:4 5.7 17

0 -00/ 0

C.

O,

03200 /0.

E0. ~~~~~~d

100- 14C-pa~titC acidstandard

1 5 10 15 20Tube No.

FIGURE 1 Countercurrent distribution of 'Cl-fatty acidmetabolites (in vivo labeling) together with added standard"C-palmitic acid.

then (Ar+i/Ar)/(Br+il/Br) = Ka/Kb. Since the rightside is constant, so must be the left. Calculation from thedata used to plot the curves in Figs. 1 and 2 shows thatthis ratio varies with successive pairs of adjacent tubes.The chlorinated lipid is therefore heterogeneous.

Urea adduction. A portion of the methyl ester mix-ture containing 'Cl-esters and "4C-methyl palmitate wasretained from the last 10 tubes of the countercurrentdistribution. Solvents were evaporated and the estersdissolved in hot 15% urea in methanol. Urea adductswere allowed to precipitate overnight at 250C. Adductedesters in the precipitate and excluded esters in the super-natant were recovered. Table II shows the isotope ratioin the original mixture, in the adduct, and in the super-natant. The chlorinated metabolites are clearly mainlybranched-chain lipids.

2500

o1875-

01. 36CI-methyl esters of I' \1250-fatty acid metabolitesad s0.

C

o625 /

14C-methyl pamte&__~~ ~ ~~- standard

5 10 15 20,Tube No.

FIGURE 2 Countercurrent, distribution of methyl esters of-'Cl-fatty acid metabolites (in vivo labeling) together withadded standard methyl 1'C-palmitate.

Lipid Metabolites of Carbon Tetrachloride 205

TABLE I IComparison of Urea Adduction of 36CI-Fatty Acid

Methyl Esters with '4C-Methyl Palmitate

36CI/14CLipid mixture 36C1 14C ratio

cOm cPmStarting mixture 2730 1561 1.7Precipitate (urea adduct) 610 1461 .42Supernatant (excluded esters) 2107 0 o0

Acetone-soluble liver lipids were likewise hydrolyzed,then analyzed by countercurrent distribution and ureaabduction. Again, the radioactivity was found incorpo-rated into branched, long-chain fatty acids.

LIVER LIPIDS AFTER THE INJECTION OF DOUBLYLABELED CCL4

Fasted rats were injected with 0.5 or 1 ml of Lipomulcontaining a mixture of '4C- and 'Cl-carbon tetra-chloride. At 15 min, approximately 0.1% of the injectedradioactivity was found incorporated in liver lipids.68% of the lipid radioactivity was found in the acetone-precipitable lipids. Fatty acid methyl esters were pre-pared from the acetone-precipitable lipid for counter-current distribution.

Fig. 3 shows the countercurrent distribution of doublylabeled methyl esters. Above the distribution curve is aplot of the wCl/14C ratio in the lipid of each tube dividedby the ratio in the injected dose, the quotient multipliedby four. It is seen that the predominant route to thelabeled lipid involves the incorporation of three of thefour chlorine atoms from each molecule of CCl4, butsome lipid is formed using all the CC14 chlorine atoms.

Formation of doubly labeled lipids by liver slices

Slices weighing 100-150 mg were taken from liversof starved rats. Two slices per flask were incubated at

WCi 4ratio of

V incorporated 2

_ 125atoms

Q)

2 100 36Ci ".

725-- -

50

5 10Tube No.

15 20

FIGURE 3 Countercurrent distribution of methyl esters ofdoubly labeled fatty acid metabolites (in vivo labeling).

TABLE I I IIncorporation of 36CI Radioactivity into Lipid by Rat Liver

Slices after Incubation in Radioactive CCd4

Time 36Cl

min cpm/mg wet tissue

5 15815 32630 48060 111460 (control slices) 4660 (control slices) 44

37°C in 3.5 ml Krebs-Ringer bicarbonate buffer towhich 0.5 ml Lipomul containing "doubly-labeled" CC14was added. Slices and buffer in two control flasks wereheated 1 min in a water bath at 100'C before theLipomul was added. Incubations were stopped after 5,15, 30, and 60 min. Control flasks were incubated 60min. After incubation, slices were removed with forceps,rinsed copiously with saline, and the lipids were ex-tracted. Table III shows the increasing incorporation ofradioactivity into slice lipids with time in the survivingslices. The extracts were then pooled, fatty acid methylesters again prepared from the acetone-precipitablelipid, and distributed in the countercurrent systemheptane-acetonitrile. Fig. 4 shows that the labeled lipidsbehave similarly to those obtained in vivo.

Free radical addition of carbon tetrachloride tomethyl oleateMethyl oleate (250 mg), carbon tetrachloride (3.5 ml)

containing '4C- and 'Cl-tracer, and dibenzoyl peroxide(7 mg) were heated together under argon in a sealedampoule 16 hr at 850C. The ampoule was then heated1 additional hr at 110'C to destroy the peroxide. Aftercooling, the reaction mixture was washed first with 10%

WC 4F -ratio of

75 incorporated 2atoms

a)o 125

a) 00 36CKi/100D75

Io-d

E 50 d"l4CCIL, 0

Tube No.

FIGURE 4 Countercurrent distribution of methyl esters ofdoubly labeled fatty acid metabolites (in vitro labeling).

206 E. Gordis

CI/C 4 X8X:XXratio ofM

V incorporated 20L atoms

625

0..

0.~~~~~~~~~~ ./I_ 5 10152

0.I

E 4250-

125-

Tube No.FIGuRE 5 Countercurrent distribution of products of per-oxide-catalyzed addition of doubly labeled carbon tetra-chloride to methyl oleate. Solvent system: heptane-aceto-nitrile, 10 ml upper phase, 10 ml lower phase.

NaHCO3, then with water. The solvent was evaporatedand the residue dissolved in petroleum ether (bp 60°-70° C). The solvent was re-evaporated, and the lipid

Methyl Final Ureaoleate reaction super- pr

mixture natant

radioactivity assayed. Approximately 3% of the radio-activity was incorporated into lipid.

A portion of the lipid residue was analyzed by counter-current distribution. Fig. 5 shows the results. The radio-active lipid behaves as long-chain ester. Under the con-ditions of this reaction (excess carbon tetrachloride),most of the product is seen to contain all four of the CCl1chlorine atoms.

Another portion of the lipid in the final reaction mix-ture was dissolved in hot 15% urea in methanol, and theadducts were allowed to form overnight at 250C. Sam-ples of the original methyl oleate, of the lipids in theampoule at the end of the reaction, of the adducted esters,and of the excluded esters were analyzed by thin-layerchromatography on AgNO3-silicic acid. The results(Fig. 6) demonstrated that a saturated product was pro-duced, and that this product was branched. On a sec-ond AgNO3-silicic acid plate, about 50 mg of the finalreaction mixture was applied to the origin as a band,and the chromatography repeated. Bands correspondingto the saturated esters and the monoenes were scrapedfrom the plate with a razor blade, and the esters eluted

FIGURE 6 Analysis of peroxide-cata-lyzed addition of labeled CC14 tomethyl oleate. Thin-layer chromatog-raphy on silver nitrate-impregnatedSilicagel G. Solvent: reagent gradechlorof orm, used without further puri-fication. Plate sprayed with dichloro-fluorescein, photographed in uv light.Samples analyzed were methyl oleate,total lipid residue at the conclusionof the reaction, the lipids in the resi-

U rea due which did not form urea adducts,re ci pita te and those that did. The products of the

reaction are seen to be saturatedbranched compounds.

Lipid Metabolites of Carbon Tetrachloride 207

from the scrapings with ethyl ether. Radioactivities ofboth fractions were assayed. 97% of the radioactivitywas found in the saturated esters.

DISCUSSION

These studies confirm Reynolds' observations thatliver lipids are labeled both by "4C- and 'Cl-carbon tetra-chloride. More than half the labeling occurs in the phos-pholipid fraction. The studies reported here show thatthe new compounds are a heterogeneous group ofbranched long-chain fatty acids containing both carbonand chlorine. Surviving liver slices can also form thesemetabolites. In a simple chemical system which gen-erates trichloromethyl free radicals, the addition ofcarbon tetrachloride to methyl oleate produced a satu-rated product which behaved like the metabolites duringurea adduction and countercurrent distribution. The evi-dence suggests that the free radical mechanism is theroute to the metabolites formed in vivo.

Any theory which relates the toxicity of carbon tetra-chloride to its metabolism must satisfy at least two re-quirements: (a) it must explain the decreasing orderof toxicity of CCl4, chloroform, and CCl3F, and (b) themetabolic transformation should occur at least as soonas the morphologic change and should help explain it.Of the three halomethanes, carbon tetrachloride formsfree radicals most easily, CCl3F least (21). The rapidityand locus of labeling of liver lipid is compatible with therate of onset and locus of the morphological changesafter ingestion of a toxic dose of CCl4. The first effecton the liver demonstrable by electron microscopy is thedisruption and vesciculation of the cisternae of the coarseendoplasmic reticulum and the dispersion of ribosomalparticles (22, 23). At this stage these ribosomes areseverely incapacitated in protein-synthesizing ability(24). Presumably the disruption of the ordered struc-ture of the endoplasmic reticulum could result fromdamage to its lipid membranes.

Recknagel has proposed that the CCl3 radicals initiatedestructive chain peroxidation of unsaturated lipids(25). The active methylene bridges of dienes and poly-ene fatty acids are excellent targets for radical attack(13). Recknagel found that peroxides could not be di-rectly identified in vivo, since their identification de-pends on determination of malonaldehyde, itself metabo-lized by liver. However, he was able to show increaseddiene conjugation after CCl4 administration, a reactionwhich accompanies lipid peroxidation. An alternativerole for the radical reaction is the formation of chlori-nated lipids, which may be unsuitable as membrane com-ponents. Cross-linking and polymerization of lipids con-taining unsaturated fatty acids might also result fromfree radical attack, along with peroxidation or chlorina-tion. The protective effect of certain antioxidants againstCCl4 poisoning (1, 2) is compatible with the radical

mechanism, since these compounds can function as freeradical inhibitors. Of course, evidence for a metabolicpathway is not proof that it is responsible for toxicity.The existence of the free radical mechanism seems wellestablished. Whether any of its possible consequencesaccount for the toxicity of CCl4 remains to be proven.

ACKNOWLEDGMENTSI wish to thank Mrs. Giedre Stankunas for skilled techni-cal assistance.

This work was supported by U. S. Public Health ServiceGrant AM03963-08.

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Lipid Metabolites of Carbon Tetrachloride 209