JOURNAL oF BACTERIOLOGY, Mar. 1969, p. 1018-1025Copyright © 1969 American Society for Microbiology

Vol. 97, No. 3Printed In U.S.A.

Effect of Endotoxin and Cortisone on Synthesisof Ribonucleic Acid and Protein

in Livers of Mice,THELMA F. SHTASEL2 Am L. JOE BERRY

Department of Biology, Bryn Mawr College, Bryn Mawr, Pennsylvania 19010

Received for publication 27 November 1968

The effect of cortisone and endotoxin, singly and in combination, on ribonucleicacid (RNA) synthesis in livers of adrenalectomized mice was determined. This wasaccomplished by measuring the incorporation either of inorganic 32P or of l:C-orotic acid into the RNA. Under similar conditions, the effect of these agents onthe rate of protein synthesis was examined with the use of "4C-leucine. Bacterialendotoxin was found to augment the uptake of isotope in the RNA and in the pro-tein of the liver. These reactions did not appear to be mediated via the pancreatichormone insulin, which was found to depress the incorporation of the radioactivecompounds into RNA. Cortisone increased the uptake of isotope in liver RNA butdepressed the incorporation of leucine into hepatic protein. These results indicatethat the previously observed ability of endotoxin to prevent the hormone inductionof hepatic enzymes, such as tryptophan oxygenase, is not associated with impairedsynthesis of liver RNA or protein.

The mechanism by means of which bacterialendotoxin prevents the hormonal induction ofcertain liver enzymes in mice has not been ex-plained. An in vitro assay for activity of an en-zyme fails to distinguish between the presence ofless enzyme because of impaired synthesis and areduced activity due to the presence of an inhibi-tor. In rat liver, it has been shown that hydro-cortisone not only results in greater de novo for-mation of such enzymes as tryptophan oxygenase(11) and tyrosine-a-ketoglutarate transaminase(16) but, as a possible mechanism for this effect,greater synthesis of hepatic ribonucleic acid(RNA) as well. Both effects of the hormone werejudged by the incorporation of the appropriateisotopically labeled precursor in amounts greaterin treated rats than in the corresponding controlanimals (17). Although endotoxin prevents thecortisone-initiated increase in activity of trypto-phan oxygenase and of phosphoenolpyruvate car-boxykinase (5), it has no readily detectable effecton the induction of tyrosine-a-ketoglutaratetransaminase (4). These observations indicatethat endotoxin is not, under these conditions, a

I This paper is taken from a dissertation submitted by thesenior author to the Faculty of Bryn Mawr College in partialfulfillment of the requirements for the Ph.D. degree.

2 Recipient of a Training Grant from the National Instituteof Allergy and Infectious Diseases. Present address: Departmentof Biology, St. Joseph's College, Philadelphia, Pa. 19131.

generalized hepatotoxin, since the synthesis of atleast one enzyme proceeds normally in its pres-ence. There are, nevertheless, a number of markedantagonisms between the action of endotoxin andthat of cortisone in the mammalian host. Someof the biological effects of endotoxin that are di-minished by cortisone are lethality, pyrogenicity,adjuvant action, increased nonspecific resistance,development of tolerance, etc. It was not surpris-ing, therefore, to observe an antagonistic actionof endotoxin against hormonal induction of cer-tain enzymes. Because of these relationships, itseemed valid to assess the effect of endotoxin onliver RNA and protein synthesis, particularly asit was able to influence the action of cortisone onthese processes.

MATERIALS AND METHODSEndotoxin. Heat-killed cells of Salmonella typhi-

murium strain SR-11 were prepared as described byBerry and Smythe (2). Appropriate dilutions of thestock suspension were made in isotonic nonpyrogenicsaline (Baxter Laboratories, Morton Grove, Ill.) toyield the desired weight in 0.5 ml. Injections were givenintraperitoneally.

Cortisone. Cortisone acetate was obtained as astabilized suspension, containing 50 mg per ml, fromUnited Research Laboratories, Inc., Philadelphia, Pa.Appropriate dilution with Baxter saline gave a finalconcentration of 5 mg of hormone in 0.5 ml. This

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was administered subcutaneously in the interscapularregion.

Insulin. Insulin, zinc protamine, and Ileitin, 40units per ml (Eli Lilly & Co., Indianapolis, Ind.),were diluted with Baxter saline to give a final concen-tration of 0.5 units in 0.5 ml. Injection was via theintraperitoneal route.

Isotopes. Carrier-free 32p as H3PO4 in 0.02 N HCIwas obtained from New England Nuclear Corp.,Boston, Mass. It was diluted with Baxter saline toprovide appropriate dosages in 0.5 ml, which wereadministered intraperitoneally 1 hr prior to sacrificein most experiments. Crystalline 14C-6-orotic acidfrom Nuclear-Chicago Corp. (Des Plaines, Ill.), 50,ucper vial (specific activity, 44.5 mc/mmole), was storedfrozen until used. For each experiment, the isotopewas dissolved in saline to give a final activity of 2 ,ucper 0.5 ml. This quantity was injected intraperi-toneally 45 min prior to sacrifice. Uniformly labeled14C-leucine in HCI, 50 uc per ml (specific activity,180 mc/mmole), was obtained from Schwarz Bio-Research, Inc., Orangeburg, N.Y., and was dilutedwith saline to give a final concentration of 2 ,uc per0.5 ml. This was given intraperitoneally 45 min priorto sacrifice.

Schedule of injections. Injections of test agents andisotopes were administered to individual mice at3-min intervals to insure an accurate exposure in eachanimal. Sacrifice was by cervical dislocation, andlivers were removed, weighed, frozen on dry ice, andstored in a freezer until extraction of RNA or proteinwas performed.

Animals. Only adrenalectomized mice were used.Swiss-Webster female mice, 6 weeks of age (DierolfFarms, Boyertown, Pa.), were adrenalectomized atBryn Mawr College and used in experiments utilizing32p. Six-week-old female mice, CFI-S (CarworthFarms, Inc., New City, N.Y.), were adrenalectomizedby the dealer. All animals were tested for the com-pleteness of surgery by the water-retention test ofBeatty, Bocek, and Peterson (1). Adrenalectomizedmice received 1% sodium chloride solution to drink.RNA extraction. In experiments with 32p, RNA was

extracted as radiopure mononucleotides by the hotsalt-ethyl alcohol procedure described by Feigelsonet al. (12). This consisted of homogenization of fivepooled livers in 0.14 M KCI with 0.0025 N NaOH andprecipitation with cold 50% trichloroacetic acid. Theprecipitate was washed with cold ethyl alcohol andthen extracted with boiling ethyl alcohol and boiling10%o NaCl to remove the protein. To the supernatantfluids containing nucleic acids, cold 95% ethyl alcoholwas added, which effected precipitation of the sodiumnucleates after overnight storage at 5 C. The sodiumnucleates were separated by centrifugation, deprotein-ized with 4:1 chloroform-octanol, and acidified; thenucleic acids were then reprecipitated with ethylalcohol. This residue was hydrolyzed by exposure to0.4 N NaOH overnight at 30 C. On the third day,deoxyribonucleic acid (DNA) was removed by pre-cipitation with 0.33 N alcoholic HCI. The mono-nucleotides were neutralized, and 1 ml was placed inplanchets and counted at infinite thinness in a Nuclear-Chicago windowless gas-flow counter. Another 1-ml

portion was added to 50 ml of potassium phosphatebuffer; a sample of this was quantitated for RNAafter spectrophotometric determination at 260 nm ina Hitachi spectrophotometer. Inorganic phosphatewas isolated from each sample and estimated accord-ing to the method of Fiske and SubbaRow (14).When 14C-orotic acid was employed, the RNA was

extracted by the Schmidt-Thannhauser technique(22). This involved homogenization of the liver inKCI, precipitation of the RNA with 10% trichloro-acetic acid, removal of lipid in boiling 3:1 alcohol-ether, and overnight hydrolysis of the RNA in 0.3 NKOH at 37 C. After neutralization, DNA and proteinwere removed from the hydrolysate by 5% trichloro-acetic acid precipitation. The ribomononucleotideswere estimated by reading at 260 nm in a Hitachispectrophotometer. Radioactivity was determined byplacing 1 ml of each sample in scintillation vials con-taining 15 ml of a dioxane cocktail and counting to aminimum of 10,000 counts in a Packard Tri-Carbliquid scintillation spectrometer, model 3310.

Determination of the 14C-leucine uptake by acid-insoluble protein was made according to the pro-cedure of deJong and Marsh (9). Liver homogenates,in 0.14 M KCI plus 0.0025 N NaOH, were treated with5% trichloroacetic acid; these precipitates were heatedin trichloroacetic acid for 15 min at 90 C to remove.nucleic acids. Lipids were extracted by 3:1 alcohol-ether, and the protein precipitate was dissolved in 1 NNaOH. After neutralization with 1 N HCI, the proteinswere again precipitated with 5% trichloroacetic acidand, after centrifugation, resuspended in 0.1 N NaOH.A 1-ml amount of this suspension was added to ascintillation vial containing 15 ml of a dioxane cock-tail for counting of radioactivity; another portion wasused for spectrophotometry. Determination of theamount of acid-insoluble polypeptides was estab-lished by use of the Warburg-Christian formula (8).

RESULTSIncorporation of 32p into hepatic RNA. In pre-

liminary experiments, RNA was extracted frommouse livers by the Schmidt-Thannhauser tech-nique (22) after injection of 82P Similar resultswere obtained for control mice and for mice thathad been injected with cortisone or endotoxinalone or with the two substances together. Thesedata were expressed as counts per minute permicrogram of RNA (i.e., as specific activity).Since the phosphate pool is subject to variationin laboratory animals under different experi-mental conditions (10), it became important,as Feigelson et al. have shown (12), to re-cover the liver RNA not necessarily quantita-tively but in radiopure form and to relate itsradioactivity to that of the phosphate pool. Thedata presented in Tables 1 and 2 are the result.The specific activities of RNA and of inorganicphosphate were each determined, and the relativespecific activity of the RNA (the ratio of the two

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previous values times 100) yielded the meaningfulfigures used for comparison in the two tables.

Replicate experiments on the pooled livers offour or five adrenalectomized mice, the numbernecessary to yield the amount of pure RNA re-quired for an accurate measure of radioactivity,were carried out (Table 1). Cortisone and endo-toxin each increased the apparent synthesis ofRNA to approximately the same extent. This canbe seen by comparing lines two and three inTable 1 with line one and with each other. Thetwo substances together have something of an

TABLE 1. Incorporation of 32p into hepatic RNAof adrenalectomized micea

Experimentaltreatment attime zero

Control

Cortisone, 5 mg

Endotoxin,0.0125 mg

Endotoxin +cortisone

Specific Specificactivity of activityRNA of pib

527 (5) 962 (5)377 (4) 962 (4)

834 (5) 866 (5)635 (5) 852 (5)

585 (5) 599 (5)797 (4) 981 (4)

Relativeactivity(activityX 100)

55 (5)39 (4)

96 (5)75 (5)

105 (5)81 (4)

1,189 (5)1934 (5)1128 (5)889 (4) 847 (4) 105 (4)

a Each value is the result obtained with pooledlivers from four or five mice. Saline (for controlmice) and cortisone were injected subcutaneously,and endotoxin was given intraperitoneally; 3hr later and 1 hr before sacrifice, 10 Ac of the iso-tope was injected intraperitoneally. The twosets of values for each treatment represent repli-cate experiments, and the numbers in parenthesesshow the number of livers pooled for the singledetermination.

I Inorganic phosphate.

additive effect (shown in the last line of Table 1),since the largest activity was obtained in thisgroup. The nature of the action of endotoxin inyielding these results is unknown.

Cortisone has been reported (3) to have noprotective effect against endotoxin lethality, andit failed to induce tryptophan oxygenase whenthe injection was given 4 hr after an LD50 of endo-toxin. The effect of the hormone, under theseconditions, on isotope uptake in RNA was, there-fore, determined. Five mice were injected withcortisone, five others received cortisone plusendotoxin, and endotoxin was administered to10 animals. After 4 hr, half of the last group re-ceived cortisone. All mice were sacrificed 8 hrafter the original injections and 1 hr after the 82phad been administered. The radioactivity of theliver RNA from these animals was calculated(Table 2). The 32p uptake by hepatic RNA in thecortisone-treated group was well above that in thecontrols, even though the elevation was some-what smaller than that observed after 4 hr (seeTable 1). Endotoxin and cortisone, when givensimultaneously, resulted in the incorporation ofthe isotope in an amount 125% above that incontrols (Table 2) and similar to that observedafter 4 hr (see Table 1). Injecting the cortisone4 hr after the endotoxin gave a result similar tothat found when the two were administered at thesame time. Thus, the 132% increase above thatin untreated animals is not greatly different fromthat found when the hormone and endotoxin weregiven together (Table 2). The failure of cortisoneto induce tryptophan oxygenase when it is given4 hr after endotoxin (4) cannot be related to ageneralized impairment in the synthesis of liverRNA. Data for mice 8 hr after they had receivedendotoxin alone were not obtained, since no micesurvived the poison for that interval of time.

Incorporation of "4C-orotic acid into hepaticRNA. The specific activity of hepatic RNA in in-

TABLE 2. Effect of an injection oJ cortisone 4 hr after endotoxin on the incorporationof lo ;MC of 32P into liver RNAa

Specific Specific Relative PercentageTreatment activity activity specific change

of RNA of P1by activity overX 100 controls

Control .. ................. 781 (5)c 1,335 (5) 59 (5)Cortisone, 5 mg..................................... 900 (5) 1,020 (5) 88 (5) 49Endotoxin, 0.0125 mg, + cortisone, 5 mg............. 1,401 (5) 1,049 (5) 133 (5) 125Endotoxin, 0.0125 mg; 4 hr later cortisone, 5 mg ... 1,138 (4) 828 (4) 137 (4) 132

a Saline in control mice and 5 mg of cortisone acetate were administered subcutaneously. Endotoxinwas injected intraperitoneally. The isotope was injected intraperitoneally 7 hr after the initial treat-ment and 1 hr before sacrifice. In one group (the last), cortisone was given 4 hr after endotoxin.

Inorganic phosphate.c Number of livers pooled for a single determination.

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dividual mice given orotic acid at intervals afterendotoxin and cortisone administration was de-termined. The RNA was extracted by the Schmidt-Thannhauser procedure (22). Time periods of 1,2, 4, and 8 hr were included in the study, and inall cases the isotope was given 45 min prior tosacrifice. The effect of delayed administration ofcortisone (4 hr after endotoxin) was also deter-mined.

In Table 3, the results obtained 1 hr after treat-ment are summarized. In each group of experi-mental animals, a numerical increase in RNAsynthesis resulted, as judged by isotope content,but the value for the group given cortisone alonewas not significantly greater than that for thecontrols. This indicates a large variation in thefour individual values that yielded the mean spe-cific activity of 609. Possibly a larger sample sizewould yield a value significantly larger than thecontrols.

In livers of mice 2 hr post-treatment, RNA syn-thesis was significantly elevated in all groupscompared with the controls. This can be seen inthe data given in Table 4. The sample sizes were

larger in this series of determinations; yet only inthe livers of mice given endotoxin did the RNAhave a specific activity that was obviously largerthan that seen after 1 hr of treatment (see Table 3).

Four hours after mice were injected with corti-sone and endotoxin, singly and combined, theincorporation of orotic acid into liver RNA wassignificantly depressed in those animals which re-ceived cortisone but was unaffected after endo-toxin alone or after the two agents combined(Table 5). The decrease that was seen in micegiven cortisone may reflect a period of reducedsynthesis that often follows an induced hyper-function. This type of "undershoot" has beenrecognized by biologists for many years and hasbeen described for a variety of physiologicalprocesses.

Table 6 presents the results obtained withlivers of mice treated 8 hr before sacrifice. In one

group, cortisone was given 4 hr after endotoxin.In every case, no significant change from thecontrol value was observed despite the fact that a

sizeable "undershoot" was evident in the mice

TABLE 3. Effect of cortisone and endotoxin, singly and combined, on the intcorporationof '4C-orotic acid in mouse liver RNAa

Experimental treatment at RA~gm) Counts per min Specific activity Percentagetime zero RNA<}g/ml)per ml x 100 change over P value

controls

Control...................... 514 (18)c 2,379 (18) 462 (18)Cortisone, 5 mg............. 367 (4) 2,193 (4) 609 (4) 32 NSEndotoxin, 5 ug .............. 408 (4) 2,593 (4) 637 (4) 38 >0.05Cortisone, 5 mg, + endotoxin,

5S,g..................... 351 (3) 2,069 (3) 585 (3) 27 >0.05

a Control mice were injected with saline and some experimental animals were injected with 5 mg ofcortisone acetate, all subcutaneously. The isotope was given 15 min after the initial injection(s) and45 min before sacrifice.

6 P values were estimated by the rank order test (24) and by Student's t test. NS = not significant.c Number of separate determinations.

TABLE 4. Effect of cortisone and endotoxin, singly and combined, on the incorporationof "C-orotic acid in mouse liver RNAa

-_________ Countsper-min Spcii aciiy PercentagebExperimental treatment at time zero RNA (,ug/ml) Counts per m Specixc ativ chage over valuecontrols

Control ..................... 514 (18)c 2,379 (18) 462 (18)Cortisone, 5 mg .............. 338 (11) 2,233 (11) 667 (11) 45 <0.05Endotoxin, 5 ,g.............. 392 (11) 3,249 (11) 861 (11) 86 <0.05Cortisone + endotoxin ....... 270 (6) 1,716 (6) 637 (6) 38 <0.05

a Control mice were injected with saline and some experimental animals were injected with 5 mg ofcortisone acetate, all subcutaneously. Other experimental animals were given 5 Ag of endotoxin intra-peritoneally. The isotope was given 75 min after the initial injection(s) and 45 min before sacrifice.

b P values were estimated by the rank order test (24).c Number of separate determinations.

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given endotoxin alone. A larger sample size mighthave rendered this finding significant.The contrast between the results in Tables 5

and 6 and those in Tables 1 and 2 merits comment.In the latter, the apparent synthesis was elevatedat 4 and 8 hr post-treatment, whereas with oroticacid either no increase was seen at these times orelse a decrease was measured. Mice from differentdealers were used in the two sets of experiments.This change was made in the course of the inves-tigation because adrenalectomized mice could bepurchased from Carworth Farms but not fromDierolf Farms. Animals from Dierolf Farms,adrenalectomized at Bryn Mawr College, wereused in the 32P experiments. Strain differencesmight have contributed to the disparity of theresults with 82p and orotic acid, but it is possiblethat the amount of istope and its location withthe RNA may have also contributed to the find-ings. The "4C-orotic acid would have been in-corporated into only the pyrimidine moiety of thenucleotides, whereas the phosphate would have

been present in all nucleotides. It is also possiblethat differences in purity of the RNA used forisotope detection contributed to these results. Theneed for caution in work of this type is madeevident by the apparent contradictions containedin the data of Tables 1 and 2 versus those ofTables 5 and 6.

Effect of insulin on "4C-orotic acid incorporationinto liver RNA. Since the pancreatic hormoneinsulin has been reported to increase amino aciduptake by muscle protein in rats (25) and hasbeen found capable of inducing hepatic trypto-phan oxygenase and tyrosine transaminase in ratliver (15), the contribution this hormone mightmake to orotic acid incorporation into liver RNAwas determined. It was found that 2 hr after theinjection of 0.5 unit of insulin and 45 min afteradministration of 14C-orotic acid no significantchange in the specific activity of liver RNA oc-curred. The mean value of five determinationswas 359, compared with 462 in 18 control mice.When endotoxin (5 ,ug) and insulin (0.5 unit)

TABLE 5. Effect of cortisone and endotoxin, singly and combined, on the incorporationof 14C orotic acid in mouse liver RNAa

Experimental treatment at time zero RNA (ug/ml) Counts perml Specific

Pcheangeor P vaperml X ~~~~~controls

Control ..................... 514 (18)c 2,379 (18) 462 (18)Cortisone, 5mg ............. 496 (9) 1,750 (9) 350 (9) -24 <0.05Endotoxin, 5Sg.............. 458 (10) 2,434 (10) 544 (10) 18 NSCortisone + endotoxin....... 484 (10) 2,211 (10) 458 (10) I NS

aControl mice were injected with saline and some experimental animals were injected with 5 mg ofcortisone acetate, all subcutaneously. Other experimental animals were given 5, g of endotoxin intra-.peritoneally. The isotope was given 195 min after the initial injection(s) and 45 min before sacrifice.bP values were estimated by the rank order test (24). NS = not significant.c Number of separate determinations.

TABLE 6. Effect of cortisone and endotoxin, singly and combined, on the incorporationof 14C-orotic acid in mouse liver RNAa

Exerimental treatment at time zero RNA (jig/ml) Counts per m Specixi chage over P valueperm X ~~~~~~controls

Control ..................... 514 (18)c 2,379 (18) 462 (18)Cortisone, 5 mg.............. 576 (4) 2,243 (4) 390 (4) -15 NSEndotoxin,5.Sg .............. 536 (4) 1,997 (4) 368 (4) -20 NSCortisone, 5 mg, + endotoxin,

5,ug ...................... 515 (4) 2,430 (4) 514 (4) 11 NSEndotoxin, 5 ;g, + cortisone,5 mg, 4 hr later............ 536 (5) 3,172 (5) 591 (5) 28 NS

a Control mice were injected with saline and some experimental animals were injected with 5 mg ofcortisone acetate, all subcutaneously. Other experimental animals were given S pg of endotoxin intra-peritoneally. In the last group of animals, cortisone was given 4 hr after endotoxin. The isotope wasgiven 435 min after the initial injection(s) and 45 min before sacrifice.

b P values were estimated by the rank order test (24). NS = not significant.c Number of separate determinations.

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were given at the same time, the mean specific ac-tivity of the liver RNA was 402. This is not sig-nificantly lower than the controls, but the in-crease produced by endotoxin alone (meansspecific activity of 861 for 11 values) was pre-vented.

Incorporation of '4C-leucine into hepatic protein.To determine whether correlative changes weretaking place in protein synthesis in livers of micein which endotoxin was producing augmentedrates of isotope incorporation into RNA, the fol-lowing studies were made. Under the same condi-tions used in measuring isotope uptake intoRNA, the incorporation of '4C-leucine into acid-insoluble polypeptides was measured (Table 7).Cortisone significantly lowered protein synthesis,as judged by this criterion, whereas endotoxinsignificantly elevated it. An injection of bothcortisone and endotoxin resulted in an "algebraicsummation" of the two opposing effects andyielded an uptake of isotope into the liver proteinthat was not significantly different from that ofthe controls (Table 7). All values were deter-mined 2 hr after the initial injections had beenmade and 45 min after the isotope. No other timesequence was employed in these experiments, so,conceivably, under other experimental conditionscontrary results might have been obtained. Themajor contradiction is the inhibition of proteinsynthesis that follows an injection of cortisone ata time when RNA synthesis is increased (compareTable 4).

DISCUSSIONThe suppression of tryptophan oxygenase ac-

tivity by endotoxin (3-5), as seen against a back-ground of augmented isotope uptake into liverRNA and liver protein, may be considered inseveral ways. (i) Genetic repression of a singleenzyme might occur in the midst of a generalized

induction of protein. If this were true, its effectwould be masked. (ii) Endotoxin, acting as an

antigen, could give rise to nonspecific and specificantibodies while, at the same time, inhibitingselected enzyme systems. (iii) Endotoxin mayeither act as an allosteric effector or, more proba-bly, result in the release of an effector(s) thatproduces sequelae of induction or suppression,or both, on different target cells or on specificmetabolic processes within cells. (iv) Endotoxinmay change certain precursor pools which mayin turn give rise to one of the aforementionedconsequences. (v) The general augmentation insynthesis of liver RNA and protein may lead tothe production of substances which elicit specificfeedback inhibition of tryptophan oxygenase.The induction of tryptophan oxygenase by

cortisone is known to depend on new proteinsynthesis (13). Accordingly, one might anticipate,a priori, that endotoxin prevents this inductioneither by blocking the action of the specificmessenger RNA required for that protein, or bydepressing RNA synthesis in general. The data inthis report do not permit a decision on the possi-bility that endotoxin inactivates specific messen-gers, but they make the postulate of a generalizedsuppression of RNA synthesis unacceptable asthe basis of endotoxin's effect on specific enzymesynthesis.

Recent studies by Bonner et al. (6) offer strongevidence for genetic derepression as a mode ofhormonal regulation. Direction interaction ofendotoxin with chromosomal material would haveto be established before this type of action wouldbe applicable to the bacterial poison. It is noteasy, moreover, to reconcile the fact that corti-sone increases RNA synthesis in an organ (theliver) where protein synthesis is also suppressed.These gaps in our understanding must be bridged

TABLE 7. Effect of cortisone and endotoxin, singly and in combination, on the incorporationof 2 &c of "4C-leucine into acid-insoluble polypeptides of livera

Proten(u/Conts er mn Spcifi actvity PercentageExperimental treatment at time zero sample) Counts permp Specific activity change over P valuebsample)per sample X 100 ~~~controls

Control...................... 2,945 (9)c 7,416 (9) 252 (9)Cortisone, 5 mg.............. 2,657 (11) 5,595 (11) 210 (11) -16 >0.05Endotoxin, 5 yg.............. 1,572 (11) 5,185 (11) 330 (11) 31 >0.05Cortisone, 5 mg, + endotoxin,

5 jug ....................... 2,006 (9) 5, 312 (9) 265 (9) 5 NS

a Control mice were injected with saline and some experimental animals were injected with 5 mg ofcortisone acetate, all subcutaneously. Other experimental animals were given 5 ,ug of endotoxin intra-peritoneally. The isotope was given 75 min after the initial injection(s) and 45 min before sacrifice.Protein was determined by absorption at 260 and 280 nm.

Pp values were estimated by the rank order test (24). NS = not significant.c Number of separate determinations.

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before satisfactory explanations are possible.Nevertheless, it is impossible to escape the con-clusion that a high degree of specificity is in-volved in the mode of action of the adrenocorti-coids and of bacterial endotoxin.RNA derived from rat spleen and lymph nodes

after immunization with Haemophilus pertussishas been reported (19) to increase template ac-tivity for amino acid incorporation when it isadded to an in vitro system derived from Esche-richia coli. This sequence of events may underliethe experiments summarized by the. data con-tained in Tables 6 and 7, where endotoxin mayhave been acting as an antigen stimulating anelevation of isotope incorporation into RNA andan increased uptake of "4C-leucine into proteinlikeproducts. An analysis of the latter, under suitablein vitro conditions, is experimentally feasible andwould provide insight into the nature of the poly-peptide which is more rapidly anabolized afterendotoxin treatment. In this connection, theabsolute amounts of RNA and protein synthesisare altered by endotoxin. Rowley (21) has shownthat cells which have phagocytosed endotoxinundergo rapid division and thereby produce alarge population of new cells, an occurrencewhich would suggest the need for an augmentedRNA synthesis to code for new cellular proteins.

Permeability changes, perhaps as a consequenceof the incorporation of endotoxin directly intothe nuclear or cytoplasmic cell membrane(Shands, personal communication), could lead toa nongenetic depression or stimulation of variousenzyme pathways. Endotoxin has been reported(23) to render hepatic lysosomal membranesmore labile than those in control rats, with therelease of proteolytic enzymes. Conversely, corti-sone, according to Merkow et al. (20), stabilizessuch membranes of alveolar macrophages inmice. How these alterations in permeability affectRNA and protein synthesis remains unclear.The suppression of tryptophan oxygenase by

endotoxin in the presence of a net increase in therate of hepatic RNA and protein synthesis may,as suggested above, result from some kind of feed-back inhibition. Cho-Chung and Pitot (7), in anexamination of the effects of breakdown productsalong the tryptophan pathway, found that re-duced nicotinamide adenine dinucleotide phos-phate and, to a lesser extent, reduced nicotinamideadenine dinucleotide inhibit tryptophan oxyge-

nase. These compounds are essentially terminalproducts of the series of reactions initiated bytryptophan oxygenase. If endotoxin affects this or

other metabolic processes, either directly or viasome feedback inhibition, the result would be a

reduction in tryptophan oxygenase activity.

Endotoxin is known to induce tyrosine-a-keto-glutarate transaminase (4), lysosomal phospha-tase (23), nonspecific antibody titers (18), andproliferation of the reticuloendothelial system(21). As a consequence of these biological conse-quences, some or many by-products would betheoretically capable of acting in the capacity ofa feedback inhibitor of tryptophan oxygenase. Itseems, at present, that some such cause is a morelikely explanation for prior observations (3-5)than an attack at the RNA level of action.

ACKNOWLEDGMENTS

This investigation was supported by Training Grant 2E-148 from the National Institute of Allergy and Infectious Dis-eases, by National Science Foundation grant GB-4019, and byPublic Health Service grant AI-07851 from the National Instituteof Allergy and Infectious Diseases.

LITERATURE CITED

1. Beatty, C. H., R. M. Bocek, and R. D. Peterson. 1954.Water tolerance test for functioning adrenal corticaltissue in the rat. Am. J. Physiol. 177:287-291.

2. Berry, L. J., and D. S. Smythe. 1961. Effects of bacterialendotoxin on metabolism. III. Nitrogen excretion afterACTH as an assay for endotoxin. J. Exptl. Med. 113:83-94.

3. Berry, L. J., and D. S. Smythe. 1964. Effects of bacterialendotoxin on metabolism. VII. Enzyme induction andcortisone protection. J. Exptl. Med. 120:721-732.

4. Berry, L. J., D. S. Smythe, and L. S. Colwell. 1966. Inhibi-tion of inducible liver enzymes by endotoxin and actino-mycin D. J. Bacteriol. 92:107-115.

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