JOURNAL OF BACTERIOLOGY, Dec. 1967, p. 1817-1823Copyright © 1967 American Society for Microbiology

Vol. 94, No. 6Printed in U.S.A.

Influence of Carbon Tetrachloride on InducibleLiver Enzymes and Response to Endotoxin in Micel

IRVIN S. SNYDER,2 MANJUL K. AGARWAL,3 AND L. JOE BERRYDepartment of Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania 19010

Received for publication 11 September 1967

Injection of mice with a sublethal dose of endotoxin 2 hr after administration of0.1 ml of carbon tetrachloride (CC14) killed 75% of the animals. CCl4 alone killed nocontrols. Significant protection against this effect was afforded by 5 mg of cortisoneand by 5 mg of nicotinamide adenine dinucleotide (NAD). With a larger dose ofendotoxin, cortisone did not give protection. Liver tryptophan pyrrolase activitywas lowered 2 hr after CC14 injection and reached a minimum after 17 hr. Inductionof tryptophan pyrrolase by cortisone, followed by administration of CC14, resultedin rapid loss of activity. A significant induction of tyrosine-a-ketoglutarate trans-aminase was observed in CCl4-treated mice. The induction of this enzyme by corti-sone was somewhat impaired when CC14 was administered concurrently with thehormone. CC14 did not lower tryptophan pyrrolase in endotoxin-tolerant mice after4 hr, but at 17 hr the activity was decreased as much as in control mice. Oxidizedpyridine nucleotides were decreased 17 hr after administration of CC14. This loss wasprevented by administration of NAD (5 mg) or by cortisone (5 mg). Carbon clear-ance from blood was reduced after treatment with CC14. These results indicate adegree of similarity between the metabolic effects of endotoxin and CCl4 in thatboth depress tryptophan pyrrolase and prevent its induction by cortisone, and bothinduce tyrosine transaminase in intact mice. Both substances appear to exert theseeffects through some type of mediated reaction.

Subcutaneous administration of small doses ofcarbon tetrachloride (CC14) to guinea pigs rendersthem abnormally sensitive to the lethal effects ofbacterial endotoxins (13-16). Associated with thisis a lowering in rate of clearance of intravascu-larly administered colloidal carbon and a subse-quent hepatic necrosis (14). CC14 is also known todisrupt a number of enzymes of the tricarboxylicacid cycle in rat liver mitochondria when thechemical is administered via a stomach tube (11).Smuckler et al. (25) have shown that the ad-ministration of CCl4 to rats results in a wide-spread dislocation of ribonucleoprotein particlesfrom the membranes of the rough endoplasmicreticulum. Associated with this is a depressedcapacity of liver microsomes from CCl4-poisonedanimals to incorporate amino acids under in

1Presented at the Third Annual Meeting of theReticuloendothelial Society, November, 1966, Be-thesda, Md.

2Present Address: Department of Microbiology,University of Iowa, Iowa City. This work was per-formed during the tenure of a research professorshipfrom the University of Iowa.

3 Present Address: Children's Hospital, Buffalo,N.Y. 14222.

vitro conditions. There appears to be, therefore, ageneralized inhibition of protein synthesis (24)resulting from CCl4 intoxication.

Berry and Smythe have presented evidence tosuggest that cortisone protection against endo-toxin lethality in mice may be related to themaintenance of hormonally inducible liver en-zymes as reflected by measurements of tryptophanpyrrolase activity (6-8). It was decided to deter-mine whether CCL4 administration influences theactivity of mouse liver tryptophan pyrrolase,another inducible liver enzyme, tyrosine-a-keto-glutarate transaminase, and those enzymes in-volved in gluconeogenesis that are believed to be,at least in part, responsible for the storage ofglycogen after cortisone administration (27, 28),and to relate these observations to endotoxinlethality. This is important because recent evi-dence obtained in this laboratory indicates thatthe reticuloendothelial system plays a metabolicrole under these conditions (1, 8). In addition,an evaluation of the activity of inducible liverenzymes should augment the evidence for theaction of CC14 as an inhibitor of protein synthesis.

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MATERIALS AND METHODSMice. Female Swiss-Webster mice (Dierolf Farms,

Boyertown, Pa.) weighing 24 -+ 1 g were used through-out the experiments. Animals were housed 10 per cage,with white pine shavings as bedding, in an animalroom held at 25 i1: 2 C. An automatic switch gave 12hr light and 12 hr darkness. Upon arrival from thedealer, mice were given a tetracycline antibiotic(Polyotic, American Cyanamid Co., Princeton, N.J.)in their drinking water for 3 days. They were placedon tap water for at least 48 hr before they were usedexperimentally. This treatment was used to increasereproducibility of results and seemed to have noundesirable side effects. Pathogen-free mouse food(D and G, Price-Wilhoite Co., Frederick, Md.) andtap water were available ad libitum unless indicatedotherwise.

Endotoxin. Heat-killed cells of Salmonella typhi-murium, strain SR-11, suspended in nonpyrogenicsaline (Baxter Laboratories, Morton Grove, Ill.),were used in all experiments. Doses were proportionalto the LD5o as calculated by the method of Reed andMuench (22). One LD5o (approximately 0.75 mg, dryweight) was given intraperitoneally in 0.5 ml of thesuspension and contained about 109 cells.

Production of tolerance. Mice were made tolerantto the crude S. typhimurium endotoxin by a scheduleof daily intraperitoneal injections. The animals re-ceived 0.1, 0.1, 0.2, 0.2, 0.4, and 0.4 LD5o, each con-tained in 0.5 ml of suspension, on successive days.These animals were used experimentally 48 hr afterthe last injection.

Injections. Isotonic, nonpyrogenic saline was usedas diluent for all injectable substances.

Cortisone acetate (United Research Laboratories,Philadelphia, Pa.), in stabilized colloidal suspension,was injected subcutaneously into the interscapularregion in 5-mg amounts in a volume of 0.5 ml.

Nicotinamide adenine dinucleotide (B-NAD:Sigma Chemical Co., St. Louis, Mo.) was injectedintraperitoneally in 5-mg amounts, in a volume of0.5 ml.

Carbon tetrachloride (Merck and Co., Inc., Rah-way, N.J.) was injected subcutaneously into theinterscapular region in 0.1-ml amounts.

Carbon clearance. The method of Benacerraf andSebestyen (2), with slight modification, was used asdescribed previously (8). Blood samples were drawnfrom the retro-orbital plexus at 5 and 10 min, and thephagocytic index k was calculated for each mouse.Liver and spleen were weighed separately on a triplebeam balance and mice were weighed on a TaconicFarms mouse scale.

Tryptophan pyrrolase assay. The technique of Knoxand Auerbach (20), as adapted to mice by Berry andSmythe (6), was used in all experiments. The cofactor,hematin, was added to each flask to convert theapoenzyme to holoenzyme as described by Eaves andBerry (Federation Proc., p. 563, 1964). Enzyme ac-tivity is expressed as micromoles of kynurenine pergram (dry weight) of liver per hour. Animals were

deprived of food from zero time until the end of theexperiment, but water was continuously available.Any turbidity in the samples to be read in the spectro-

photometer was removed by centrifugation in poly-ethylene tubes at 12,000 X g for 15 min at 20 C.

Tyrosine-a-ketoglutarate transaminase assay. Themethod of Rosen and Milholland (23) was used. Theenzyme activity is expressed as micrograms of p-hydroxyphenyl pyruvic acid (PHPP) per gram (dryweight) of liver per 10 min.

Assay for pyridine nucleotides. The method ofKaplan et al. (17) was followed for quantitative deter-mination of the oxidized pyridine nucleotides con-tained in a 5% trichloroacetic acid extract of liver. ATurner fluorometer with primary filter, no. 7-60 (360m,u exciting light), and a secondary filter, no. 75 (490mA transmitted light), was used to measure the fluores-cence of the stable compounds that form from thenucleotides in the presence of hot 5 N NaOH. Addi-tional details are given in an earlier publication (6).

Determination of liver glycogen. Liver glycogen wasmeasured by the method of Kemp and Kits vanHeijningen (18), with the Coleman model 14 spectro-photometer.

Survival experiments. Mice were challenged withproportional doses of LD5o of endotoxin and the num-ber of survivors after 48 hr was recorded. No food wasgiven during this period, although water was available.

Statistics. The significance of difference in the meanresponse between treatments was determined by the ttest. The rank order test of Wilcoxon (30) was used todetermine the significance of differences between sur-vivorship after various treatments.

RESULTS

CCl4 and susceptibility to endotoxin. Intra-peritoneal injection of mice with 0.0625 LD50 ofendotoxin 2 hr after subcutaneous administrationof 0.1 ml of CCl4 killed 15 of 20 animals (25%survival), but no animals died when given eithersubstance alone (100% survival). Results areshown in Table 1. Injection of cortisone or nico-tinamide adenine dinucleotide (NAD) at the timethat endotoxin was administered resulted insignificant protection of the animals (90 and65% survival, Table 1). Groups of mice givenCC14 and increasing amounts of endotoxin(0.125, 0.25, 0.5, 1.0 LD5o for normal mice) wereprogressively susceptible, as indicated by reduced

TABLE 1. Influence of CC14 alone or in combinationwith cortisone or nicotinamide adenine dinucleotide

(NAD) on endotoxin lethality in mice

Injection Living/ Suir- Significanceinjectedvia

1. CC14 + endotoxin (1/16 5/20 25LDs0)

2. CCl1 + endotoxin + 18/20 90 2vs. 1 P < 0.008cortisone (5 mg)

3. CC14 + endotoxin + 13/20 65 3 vs. 1 P < 0.028NAD (5 mg)

4. Endotoxin (1/16 LD6o) 20/20 1005. CC14 (0.1 ml) 20/20 100

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CC14 AND RESPONSE TO ENDOTOXIN

survival times when compared with controlgroups. These data are not shown, but the differ-ences in survivorship were smaller as largeramounts of endotoxin were used and fewersurvivors remained.These results should be compared with those

obtained when a larger dose of endotoxin was

employed. An amount of endotoxin that per-mitted 70% survival when given alone and 20%survival when given after CC14 could not beprotected against by cortisone when the hormonewas given in combination with the CC14 (Table2). This result was different from that shownin Table 1. Cortisone, when given with endotoxinbut without the CCl4, gave 100% survival, as didCC14 alone (Table 2). One must conclude, there-fore, that the complex interaction between CCl4,endotoxin, and cortisone cannot be generalized,but is clearly dose dependent.

Influence of CC14, cortisone, and NAD on levelsof tryptophan pyrrolase and oxidized pyridinenucleotides. Because mice were protected againstthe combined effect of CCl4 and a normally sub-lethal dose of endotoxin by administration ofNAD or cortisone (Table 1), measurements ofoxidized pyridine nucleotides and tryptophanpyrrolase were carried out on livers of mice 17 hrafter injection of CCl4, cortisone, and NAD,singly and in combination. These measurementsare important, because tryptophan pyrrolase isbelieved to initiate the sequence of reactionsresponsible for the conversion of tryptophan intonicotinamide, a constituent ofNAD and nicotina-mide adenine dinucleotide phosphate (NADP),and because of the relationship that may existbetween the levels of NAD and the survival ofendotoxin-poisoned mice (6, 7). The level ofoxidized pyridine nucleotides was below that ofthe controls (Table 3) 17 hr after CC14. An injec-tion of 5 mg of cortisone raised the nucleotidecontent of liver, after 17 hr, above that of the

TABLE 2. Influence of CCI4 alone and in combinationwith endotoxin and cortisone on lethality in micel~~~~~~

Injection iLiving! Sur.- Significanceinjected vival

1. Endotoxin (0.5 LD5o) 14/20 702. CC14 + endotoxin 4/20 20 2 vs. 1 P < 0.0083. CC14 + endotoxin + 5/20 25 2 vs. 3 P N.S.a

cortisone (5 mg)4. Endotoxin + cortisone 20/20 100 4 vs. I P < 0.015. CC14 20/20 100

a N.S. = not significant.

control; a concurrent injection of hormone andCC14 resulted in a value significantly higher thanthat found after CC14 alone. A 5-mg amount ofNAD alone did not significantly elevate thenucleotides above the control value, but a 10-mgamount did (data not shown; a value of 1.49 ,ug/

mg of liver was obtained). The smaller dose ofNAD (5 mg), when given with CC14, showed avalue about the same as that with NAD alone,and significantly higher than with CC14 alone(Table 3).Tryptophan pyrrolase values following similar

treatment are also presented in Table 3. Theactivity was almost completely lost 17 hr afterCCl4 administration. This low value was approxi-mately doubled in mice injected concurrently witheither cortisone or NAD, but it was well belowthat found in control animals, or especially inanimals given cortisone alone, where the activitywas more than twice that of the controls. Thus,protection of CCl4 pretreated mice against endo-toxin lethality by means of cortisone or NADadministration did not correlate well with a main-tenance of tryptophan pyrrolase activity, but theamount of oxidized pyridine nucleotides seemedto show slightly better correlation, as judged bythe one time at which assays were made. We are

TABLE 3. Pyridine nucleotide and tryptophan pyrrolase 17 hr after each treatment

Treatment Pyridine nucleotide Significance Tryptophan Significance(,ug/mg of liver) pyrrolasea infiac

1. Control 0.61 0O.02b (17)c 22.9 i4 3.2 (8)2. CCI4 (0.1 ml) 0.44 i 0.03 (18) 2 vs. 1 P < 0.001 3.1 :1: 0.6 (8) 2 vs. 1 P < 0.0013. Cortisone 0.70 ± 0.04 (10) 3 vs. 1 P = 0.001 54.4 -4 12.5 (11) 3 vs. 1 P < 0.0014. CCI4 + cortisone 0.53 ± 0.03 (8) 4 vs. 2 P = 0.01 6.1 i 1.0 (8) 4 vs. 2 P < 0.015.NADd (5 mg) 0.68 ± 0.15 (9) 5 vs. 1 N.S.e6. CC14 + NAD (5mg) 0.72 i 0.08 (10) 6 vs. 2 P = 0.01 5.3 ±t 1.4 (8) 6 vs. 2 P < 0.001

a Micromoles of kynurenine per gram (dry weight) of liver per hour.b Standard error of the mean.c Number of determinations, one determination per mouse.d NAD = nicotinamide adenine dinucleotide.e N.S. = not significant.

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not certain that the small, but statistically signifi-cant, rise in tryptophan pyrrolase in mice givenboth CC14 and cortisone or NAD played a role inprotection.

Influence of CC14 on kinetics of tryptophanpyrrolase induction. Mice were injected with CC14alone, cortisone alone, or with CC14 2 or 13 hrafter an injection of cortisone, and the activity oftheir liver tryptophan pyrrolase was followed withtime (Fig. 1). Mice receiving CC14 alone showed aloss in enzyme activity after 2 hr and a minimallevel after 12 to 24 hr. Injection of cortisone alonegave a typical induction curve similar to thatreported previously (9). The enzyme activity inlivers of mice given CC14 2 hr after cortisonedecreased in a manner that essentially paralleledthe curve obtained with CC14 alone. The minimalvalue was reached at 17 hr, and this was followedby a slight increase at 24 hr. The enzyme levelwas not back to normal after 36 hr.When CC14 was injected 13 hr after cortisone, a

time when the level of tryptophan pyrrolase isabout ready to decrease, activity diminished morerapidly than in control animals. This suggestseither an accelerated destruction of enzyme or amore prompt drop in rate of synthesis.

CC14 and tyrosine-ca-ketoglutarate transaminase.The activity of liver tyrosine-a-ketoglutaratetransaminase is known to increase in mice afteran injection of endotoxin (9). Measurement ofthis enzyme 17 hr after treatments of the typedescribed above revealed significant inductionafter the administration of CCl4 alone, and evenafter both CC14 and cortisone were given (Table 4).In both cases, however, induction was less thanwith cortisone alone. This suggests that CC14,

6C

30

It

) *~~~~~~~~~~-ACC14CCI4 Injected a-" Cortisone acetate

and CCI4. \. \-. Cortisone ocetate

CCI/14 \ \ *--CCI4 injectedCCI4 \ \ " 13 brs fter cartisane

Injected.

Vs

A

O 4 8 12 16 20 24 28 3Z 36TIME OF ASSAY(hr)

FiG. 1. Changes in mouse liver tryptophan pyrrolaseactivity (pnoles ofkynurenine per g dry weight ofliverper hr) with time. Some animals were given a subcu-taneous injection of 5 mg of cortisone acetate at minus2 hr, followed with time (0); others were then injectedsubcutaneously with 0.1 ml of carbon tetrachloride(CC14) 2 hr (0) or 13 hr (dashed line) later. Mice givenCC!, alone at time zero, A.

TABLE 4. Effect of CCI4 and cortisone on tyrosine-a-ketoglutarate transaminase activity

in mice"

Experimental Tyrosine-a-ketoglu+a-treatment rate transaminasea Signficance

1. Control 14.9 ± 0.9C (18)d2. CC14 (0.1 20.7 ± 1.9 (12) 2 vs. 1ml) P < 0.001

3. CC14 + 29.3 ± 1.9 (12) 3 vs. 1cortisone P < 0.001(5 mg)

4. Cortisone 40.3 ± 2.7 (12) 4 vs. 2(5 mg) P < 0.001

4 vs. 3P < 0.001

a Assays were done 17 hr after injection.b Micrograms ofparahydroxyphenyl pyruvic acid per

mg (dry weight) of liver per 10 mi.C Standard error of the mean.d Number of determinations, one determination per

mouse.

TABLE 5. Effect of CCI4 addition in vitro on livertryptophan pyrrolase and tyrosine-a-ketoglutarate transaminase activity"

Tryptophan pyrrolase Tyrosine-a-ketogluta-Homogenate rate transaminase

no.Control +CCh Control +CC1

1 0.246 0.273 0.313 0.3352 0.270 0.286 0.299 0.3023 0.188 0.197 0.382 0.3774 0.571 0.600 0.291 0.2885 0.321 0.368 0.286 0.2926 0.299 0.285

* Results are given in optical density readings.

possibly because ofitsknownhepatoxic properties,is able to suppress the full hormonal inductionof tyrosine-a-ketoglutarate transaminase.

In vitro effect of CC4 on liver homogenates. Todetermine whether the action of CC14 in loweringtryptophan pyrrolase and in partially suppressingtyrosine-a-ketoglutarate transaminase inductionis due to a direct inactivation of the enzymes or toan indirect (mediated) effect, 0.1 ml of CC14 wasadded directly to homogenates of liver tissueprior to the incubation period required for theenzyme assays. Comparison of optical densityreadings on liver homogenates with and withoutthe addition of CC14 indicated no direct effect onthe assay for either tryptophan pyrrolase ortyrosine-a-ketoglutarate transaminase (Table 5).

Effect of CC!4 on carbon clearance. Mice in-jected with 0.1 ml of CC14 showed a significantlydepressed rate of carbon clearance (k values) after

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TABLE 6. Influence of CCl4 on blood clearance of carbon in mice

Experimental treatment Phagocytic index k value Significance

1. Control 0.048 4 0.007a (8)b2. 4 hr after 0.1 ml of CC14 0.030 i 0.006 (11) 2 vs. 1 P < 0.013. 17 hr after 0.1 ml of CCl4 0.011 -+ 0.002 (10) 3 vs. 1 P < 0.001

a Standard error of the mean.b Number of determinations, one determination per mouse.

TABLE 7. Changes in liver tyrptophan pyrrolaseactivity in endotoxin-tolerant mice after

an injection of either endotoxin orCC14

Liver tryptophan pyrrolase activityaExperimentaltreatment

after 4 hrs after 17 hrs

Saline ......... 26.7 4 1.3 (8)Endotoxin. ........34.3-4- 1.41 (8)c 26.7 i 2.3 (8)CC14 (0.1 ml).. 27.3 i 4.0 (8) 3.9 4 1.3 (8)

a Micromoles of kynurenine per gram (dryweight) of liver per hr.

b Standard error of the mean.c Number of determinations, one determination

per mouse.

TABLE 8. Changes in liver glycogen in mice injectedwith either cortisonie or CC14 alone or

in combiniation

Experimental Liver glycogen Significancetreatment (% by weight)

1. Control(no previous 5.1 i 0. 3a (8)binjection)

2. Cortisone (5 mg) 10.0 4i 0.5 (12) 2 vs. 1 P < 0.0013. CC14 (0.1 ml) 1.1 :410.1 (8) 3vs. I P < 0.0014. CC14 + cortisone 1.5 i 0.1 (8) 4 vs. 1 P < 0.001

a Standard error of the mean.b Number of determinations, one determination per mouse.

an interval of 4 hr and an even greater depressionafter 17 hr (Table 6). These changes were notassociated with any significant decrease in liveror spleen weight in CC14-treated mice (data notshown), so that the impaired carbon clearancemust be dependent upon a lack of phagocyticability of the macrophages.Enzyme activity in endotoxin-tolerant mice.

Challenge of endotoxin-tolerant mice with endo-toxin failed to alter significantly the activity oftryptophan pyrrolase after 4 and 17 hr. This isshown in Table 7 and confirms earlier results (8).The rise seen at 4 hr is not statistically significant,but a similar change was described previously (8).CCl4 had no effect on the enzyme 4 hr postinjec-tion, but caused a profound decrease in activityafter 17 hr.

Effect of CC14 on cortisone induced gly-coneogenesis. Mice were injected with cortisone orCCl4 singly or in combination, and their liverglycogen was assayed subsequently. The controlvalue of 5.1%, typical of livers of fed mice (5),doubled 17 hr after the administration of corti-sone (Table 8). This increase occurred eventhough the mice were denied food throughout the17-hr period postinjection. Obviously, the eleva-tion in carbohydrate under these conditions hadto result from glyconeogenesis. CCl4 alone reducedliver glycogen to 1.1 % after only 4 hr, and itprevented almost completely the glyconeogenesisthat should accompany an injection of cortisone(Table 8).

DISCUSSION

The findings described in this report are con-sistent with earlier evidence that suggests a criticalrole for enzyme induction in the response ofmammals to bacterial endotoxin (3, 6-9) and toother forms of stress (Berry, unpublished data),against which adrenocorticoids are effective. Theenzymes that seem to be most consistently in-volved in the earliest phases of the animal'sreaction to stress are tryptophan pyrrolase, tryo-sine-a-ketoglutarate transaminase, and the en-zymes that are crucially involved in glyconeogene-sis. One of them, tyrosine-a-ketoglutaratetransaminase, seems to respond differently fromthe other enzymes with short half-life. In intactmice, it is induced rather than depressed byendotoxin, by CCl4 (Table 4), by reticuloendothe-lial-active colloids (1, 4), and by hypoxia (Berry,unpublished data). Why these differences occur isunexplained, but the fact that they do is clearlyestablished.

It would be unrealistic to assume that CCl4,with its generalized toxicity and specific hepato-necrotizing action, has as its only or primaryeffect the prevention of induction of the fewenzymes under study. It has been found, however,to upset protein synthesis in general (24), pos-sibly through an alteration in liver ribosomes(25); to block glycogen synthesis in liver slices(29); to disrupt a number of enzymes in livermitochondria (11); and to inhibit the NAD-

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linked oxidation of fatty acids (14). Certaincytochrome c reductase activities have beenfound to increase in CCl4-poisoned animals (12)so that the behavior of tyrosine-a-ketoglutaratetransaminase is not unique.The one inconsistency in the present study was

the ability of cortisone to protect against endo-toxin lethality in CC14 pretreated mice (Table 1)under circumstances where tryptophan pyrrolaseactivity could not be maintained (Table 3) andwhere glyconeogenesis did not occur (Table 8).It was necessary to administer a small dose ofendotoxin, under these conditions, in order forcortisone to be protective (compare data of Table1 and 2). Thus, cortisone reduces endotoxinlethality in CCl4-pretreated mice when a smalldose is given, but with a larger amount, no pro-tection occurs. Additional experiments over awider range of time intervals and dosages wouldhave to be done to clarify these relationships.

Other workers have found that CCl4 sensitizesto endotoxin. Formal and co-workers (15) ob-served that guinea pigs are sensitized to Shigellaflexneri endotoxin by CC14 and are also mademore susceptible to oral infection by the sameorganism (16). Farrar and Corwin (14) relatedmaximal susceptibility in guinea pigs to the periodof greatest liver damage, and Farrar and Magnani(13) had reported earlier that 3 mg of cortisonegiven to CCl4-treated guinea pigs sensitized themto endotoxin rather than protecting them. Chedid(10) and Berry (3) had found it impossible toinfluence the response of guinea pigs to endotoxinby a large dose of cortisone (15 mg).The influence of CC14 on liver tryptophan

pyrrolase activity in endotoxin-tolerant mice isinteresting. Berry and Smythe (8) have shownthat administration of endotoxin to tolerantmice fails to lower liver tryptophan pyrrolaseactivity. Our data with endotoxin-tolerant micenot only confirm this (Table 7), but make itevident that the enzyme is also resistant to CC14poisoning for a period of 4 hr but not for 17 hrpostinjection. Whether the resistance of the en-zyme to CC14 at 4 hr is related to an increase inreticuloendothelial system activity resulting fromthe endotoxin injections is unknown. However,the data suggest that enzyme maintenance mayplay an important role in the resistance of tolerantmice to endotoxin. This may be the result, asseveral investigators believe (13, 14), of theability of tolerant animals to detoxify endotoxinin liver and spleen more effectively than controlanimals. Perhaps the immunological explanationof tolerance by Watson and Kim (26) suggests anenhanced removal of endotoxin, or more rapidneutralization, along lines suggested by Radvanyet al. (21), Kim and Watson (19), and others.

How this would explain the observations withCCl4 is obscure. In any case, the loss of enzyme intolerant animals injected with CC14, and main-tenance of enzyme in tolerant animals challengedwith endotoxin, show that some specificity isassociated with endotox;n tolerance.

ACKNOWLEDGMENTSThe technical assistance of Anne Sargent is grate-

fully acknowledged.This investigation was supported by National

Science Foundation grant no. GBA4019 and by agrant from Smith Kline and French, Inc., Phila-delphia, Pa.

LITERATURE CITED1. AGARWAL, M. K., AND L. J. BERRY. 1966. Effect

of RES-active agents on tryptophan pyrrolaseactivity and endotoxin lethality. J. Reticulo-endothelial Soc. 3:223-235.

2. BENACERRAF, B., AND M. M. SEBESTYEN. 1957.Effect of bacterial endotoxins on the reticulo-endothelial system. Federation Proc. 16:860-867.

3. BERRY, L. J. 1964. Effect of endotoxins on thelevels of selected enzymes and metabolites,p. 151-159. In M. Landy and W. Braun [ed.],Bacterial endotoxins, Rutger Univ. Press, NewBrunswick, N.J.

4. BERRY, L. J. 1965. The reticuloendothelial sys-tem in tolerance to bacterial endotoxins. Pro-ceedings of 4th International Symposium onRES. NISSHA Printing Co., Mibu, Nakagyo-ku, Kyoto, Japan.

5. BERRY, L. J., D. S. SMYTHE, AND L. G. YOUNG.1959. Effects of bacterial endotoxin on metabo-lism. I. Carbohydrate depletion and the protec-tive role of cortisone. J. Exptl. Med. 110:389-403.

6. BERRY, L. J., AND D. S. SMYTHE. 1963. Effects ofbacterial endotoxins on metabolism. VI. Therole of tryptophan pyrrolase in response ofmice to endotoxin. J. Exptl. Med. 118:587-603.

7. BERRY, L. J., AND D. S. SMYTHE. 1964. Effects ofbacterial endotoxins on metabolism. VII.Enzyme induction and cortisone protection. J.Exptl. Med. 120:721-732.

8. BERRY, L. J., AD D. S. SMYTHE. 1965. Somemetabolic aspects of tolerance to bacterialendotoxin. J. Bacteriol. 90:970-977.

9. BERRY, L. J., D. S. SMYTHE, AND L. S. COLWELL.1966. Inhibition of inducible liver enzymes byendotoxin and actinomycin D. J. Bacteriol.92:107-115.

10. CHEDID, L., F. BOYER, AND M. SAvIARD. 1954.Nouveaux aspects de l'action antitoxique de lacortisone. Ann. Inst. Pasteur 86:347-355.

11. CHRISTIE, G. S., ANm J. D. JUDAH. 1954. Mecha-nisms of action of carbon tetrachloride on livercells. Proc. Roy. Soc. (London) 142:241-257.

12. CLEVELAND, P. D., AND E. A. SMUCKLER. 1965.Effect of CC14, dimethylnitrosamine, andthioacetamide on hepatic DPNH and TPNH

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cytochrome C reductase. Proc. Soc. Exptl.Biol. Med. 120:808-810.

13. FARRAR, W. E., AND T. MAGNANI. 1964. Endo-toxin susceptibility following hepatic injury bycarbon tetrachloride. Proc. Soc. Exptl. Biol.Med. 115:596-600.

14. FARRAR, W. E., JR., AND L. M. CORWIN. 1966.The essential role of the liver in detoxificationof endotoxin. Ann. N.Y. Acad. Sci. 133:668-684.

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