effect of thioacetamide on adenylic acid deaminase activity and … · nuclear and cytoplasmic sap...

A specific deaminase for the conversion of adenylic acid(AMP) to inosinic acid (IMP) and ammonia was demonstrated in rat liver (22, 34) and in rat tumors of hepaticorigin (22). The specific activity of AMP deaminaseamong rat tumors of hepatic origin and in precancerouslivers of rats fed 3'-methyl-4-dimethylaminoazobenzene(3'-Me-DAB) was higher than normal rat liver (11, 22).Further studies on AMP deaminase in precancerous ratliver showed that procedures known to prevent or delaythe onset of 3'-Me-DAB induced hepatic cancers also prevented or delayed the increase in enzyme activity (23).Although it was concluded that the elevation of thisenzyme activity was related to azo dye carcinogenesis(23), the time necessary to achieve significant elevationswas too long to permit definitive studies into mechanismsunderlying these increases.

Fitzhugh and Nelson (16) reported that thioacetamidecaused hepatic cancer when fed to rats and their observa..tion was subsequently verified (18, 19). In addition tobeing hepatocarcinogenic, thioacetamide, when fed, causedincreases in the size of hepatic nuclei and nucleoli (18, 19,24, 35, 45) and increases in the amount of nuclear proteinand nuclear RNA (18, 26). On the other hand, thioacetamide ingestion caused no change in the amount ofhepatic deoxyribonucleic acid (DNA) (18, 24, 26) or inthe rate of nuclear RNA biosynthesis (18). Adams andBusch (1) found that multiple intraabdominal injectionsof thioacetamide caused increases in hepatic nuclear RNAbut suppressed the rate of nuclear RNA biosynthesis.

Received for publication November 12, 1964.

These results (1) suggested that injections of thioacetamidemight rapidly produce some of the hepatic responsesusually seen after more prolonged feeding.

Our purpose initially was to determine (a) whether thioacetamide fed under conditions which caused hepaticcancers (16, 18, 19) would cause increases in hepatic AMPdeaminase, and (b) whether injections of thioacetamidewould cause increases in AMP deaminase activity. Datafrom these experiments indicated that after 2 weeks offeeding or 3 daily injections of thioacetamide, hepaticAMP deaminase activity was significantly increased.Time-dosage studies, with a regimen of once daily thioacetamide injections, yielded data showing increases inAMP deaminase activity, diameters of nuclei and nucleoli,and the amount of nuclear RNA. Subfractionation ofnuclear RNA into â€œnon-nucleolarâ€•nuclear (NNN) andâ€œnucleolarâ€•fractions indicated that only â€œnucleolarâ€•RNAincreased in quantity ; however, the rate of orotic acid-6-'4Cincorporation into both fractions was increased.

MATERIALS AND METHODS

Animals.â€”Female rats used throughout this work werepurchased from the Holtzman Company, Madison, Wisconsrn, and at the beginning of any experimental regimenweighed 150â€”180gm. They were maintained on Rockland chow diets unless fed thioacetamide or 3'-Me-DAB.When thioacetamide was fed it was added at 2 concentrations, 0.033 % and 0.066 %, to a semisynthetic diet described by Medes et al. (30). The azo dye, 3' Me-DAB,was fed at 0.06 % in the same diet. In experiments in

596

Effect of Thioacetamide on Adenylic Acid Deaminase Activityand Nuclear Ribonucleic Acid Metabolism in Rat Liver

DONALD E. KIZER, B. C. SHIRLEY, BETTYE Cox, @ti@iBorr A. HOWELL

(Biomedical Division, The Samuel Robert8 Noble Foundation, Inc., Ardmore, Oklahoma)

SUMMARY

Thioacetamide was administered to rats to determine whether increases in hepaticadenylic acid (AMP) deaminase activity could be achieved in shorter time intervalsthan had been previously observed with azo dyes (23). Thioacetamide was added toa semisynthetic diet at 0.033 % or 0.066 % and was fed to rats for 12 weeks. Within 2weeks hepatic AMP deaminase activity was significantly elevated and remainedelevated throughout 12 weeks. When thioacetamide was injected intraabdominallyonce daily for 9 days at 50 mg/kg body weight, hepatic AMP deaminase activity wassignificantly elevated after 3 injections. This regimen also caused increases in theamount of nuclear ribonucleic acid (RNA), and in the diameters of nuclei and nucleoli.Nuclear RNA was subfractionated into â€œnon-nucleolarâ€•nuclear (NNN) and â€œnucleolarâ€• fractions and it was observed that nearly all the increase in nuclear RNAoccurred in the â€œnucleolarâ€•fraction. The rates at which orotic acid-6-'4C wasincorporated into these 2 fractions indicated that 3 injections of thioacetamide increased â€œnucleolarâ€•RNA synthesis; whereas 3, 6, or 9 injections increased NNN RNAsynthesis.

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KIZER et al.â€”Effects of Thioacetamide on Rat Liver 597

which thioacetamide or 3'-Me-DAB was fed to rats, control animals were fed the semisynthetic diet withoutcarcinogen.

Thioacetamide was injected intraabdominally once dailyat a concentration of 50 mg/kg body weight. Controlanimals received an equivalent volume of 0.9 % salineonce daily. Unless otherwise specified, animals werekilled 24 hr after the last injection. Orotic acid-6-'4Cwas diluted with saline to give a dosage of approximately3.3 Mc/2 ml, and this volume was injected intraabdominally24 hr after the last injection of th.ioacetamide or saline.Animals were killed at 30 min or 1 hr after isotope injection.

Preparation of tisst@e.â€”Animals were killed by cervicalfracture. Livers were quickly excised and then werechilled and finely minced in the appropriate homogenizingmedium.

Tissue for enzyme assays was homogenized in 0.1 Mcitrate buffer, pH 6.0, and the homogenates were centrifuged at 105,000 X g in a Spinco model L preparativeultracentrifuge for 30 min (22). The clear supernatantfluid was assayed for enzyme activity.

For experiments in which the diameters of nuclei andnucleoli were measured, the right lateral lobe of the liverwas fixed in 10 % formalin containing 1 % calciumchloride (5).

When hepatic tissue was to be analyzed for RNA bythe Schmidt-Thannhauser-Schneider method (47), it washomogenized in 0.25 M sucrose. The homogenates weredivided into 2 portions. One portion was centrifuged at105,000 X g for 30 mm and the supernatant fluid wasanalyzed for RNA (cytoplasmic sap RNA). The otherportion was filtered through glass wool pads; the nucleiwere separated by 2 centrifugations in an Internationalcentrifuge model PR-2 at 700 X g (37); and, the nucleiwere anlyzed for RNA (nuclear RNA).

When the extraction method of Allfrey and Mirsky (2)was employed for the subfractionation of RNA fromnuclei, livers were perfused with chilled 0.9 % saline andthen with 0.25 M sucrose (44). Livers were homogenizedin 0.25 M sucrose containing 0.003 M CaCl2 (3). Nucleiwere separated by 2 centrifugations at 700 X g (37). Insome experiments nuclei were purified by centrifugationin 2.2 M sucrose (13) prior to subfractionation of nuclearRNA (2).

Enzyme assays.â€”Enzyme assays were performed exactly as described previously (22). Products of theenzyme reaction were analyzed as described previously(22) except that ammonia was analyzed by the method ofChaney and Marbach (12) exclusively.

Measurement of nuclei-nucleoli diameters.â€”Fixed tissuewas imbedded in paraffin ; sections were cut at 5@ andwere stained with Schiff-methylene blue (42). Diameterswere measured under oil immersion using a microscopefitted with an ocular micrometer. In each liver 100nuclei and all of the nucleoli in each nucleus were measured.

Nuclear and cytoplasmic sap RNA .â€”RNA was separatedin isolated nuclei or in the 105,000 X g supernatant fraction of whole liver homogenates by the Schmidt-Thannhauser-Schneider procedure (47). Quantitative estimation of RNA was made by reaction with orcinol (47). The

optical density was determined at 660 m@iin a Bausch andLomb Spectronic 20 and was compared to standards prepared with yeast RNA. Aliquots of the solution afterbase hydrolysis were removed for protein determinationsby the method of Lowry et al. (27).

Subfractionation ofnuclear RNA .â€”Nuclei were extractedonce with 0.1 M potassium phosphate buffer, pH 7.1 (2).Borrowing the nomenclature employed by Koulish andKleinfeld (25), the RNA in the buffer layer is called â€œnonnucleolarâ€•nuclear (NNN) RNA throughout this report.Ribonucleoproteins in the buffer layer were precipitatedand washed in the general manner of the Schmidt-Thannhauser-Schneider procedure (47), except that the modifications of Steele et al. (44) were adopted ; i.e., ribonucleoproteins were precipitated with 0.75 N perchioric acid andwere washed with 90 % ethanol containing 2 % sodiumacetate. The ribonucleoproteins were solubilized in 1 NNaOH and aliquots were removed (a) for quantitativeRNA determination by reaction with orcinol (47) usingyeast RNA as a standard ; (b) for protein determination(27) using crystalline bovine albumin as a standard ; and(c) for determination of isotope content by plating onstainless steel planchettes and counting in a gas flowcounter fitted with a â€œMicromilâ€•window. Counts werecorrected for background and self-absorption.

The residue after buffer extraction was washed once withthe same buffer and then was extracted 1 hr with 1 MNaCl (2). The residue which remained after salt extraction was precipitated with 0.75 N perchioric acid andwashed successively with perchloric acid and ethanolsodium acetate (44). The RNA fraction obtained in thismanner is designated as â€œnucleolarâ€•RNA throughout thisreport. The ribonucleoproteins were solubilized in 1 NNaOH and aliquots for quantitative estimates and forestimates of radioactivity were withdrawn as describedin the previous paragraph.

Sources of chernicals.â€”Thioacetamide was purchasedfrom J. T. Baker Company, Phillipsburg, New Jersey,but 3'-Me-DAB was synthesized using the procedure described by Giese et al. (17). Orotic acid-6-'4C was purchased from Volk Radiochemical Company, Chicago,Illinois. AMP, IMP, and yeast RNA were purchasedfrom Pabst Laboratories, Milwaukee, Wisconsin.

RESULTS AND DISCUSSION

Fitzhugh and Nelson (16) reported that rats fed 0.1 %thioacetamide survived less than a month. Animals fed0.05 % thioacetamide survived, but showed marked nodular cirrhosis, while those fed thioacetamide at 0.025 %showed only slight or moderate cirrhosis. We addedthioacetamide to semisynthetic diets (30) at 0.033 % and0.066 % and fed these diets for 12 weeks. Hepatic AMPdeaminase activity at various intervals during 12 weeks isshown in Chart 1. Significant increases in AMP deaminase activity were observed within 2 weeks with bothconcentrations of thioacetamide and these elevationspersisted throughout the 12-week feeding period. Thehigher concentration of thioacetamide consistently eliciteda greater increase in this hepatic enzyme activity.

When compared with thiourea, thioacetamide appearedto be a relatively weak hepatocarcinogen ; however, it was



598 Cancer Research Vol. 25, June 1965

24

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4 8 12Weeks on Diet

CHART 1.â€”The effect of thioacetamide fed in a semisynthetic

diet upon the adenylic acid deaminase activity of rat liver. Liverswerehomogenizedin 0.1Mcitrate buffer,pH 6.0,and homogenateswere centrifuged 30 mm at 105,000X g. Sufficient supernatantfluid to give 1-3 mg protein was incubated with 13.3 mr@sAMP in0.1 M citrate buffer, pH 6.0, for 30 mm at 38Â°C. Each valuerepresents the average of assays on 7 animals with the exceptionof the 10-week points where 4 animals were used. The verticalline represents the S.E.

CHART 2.â€”Comparison of the effect of thioacetamide and 3'-

Me-DAB on the hepatic adenylic acid deaminase activity. Valuesplotted were calculated:

@@molesIMP/mg protein in experimental rats@pmoles IMP/mg protein in basal diet fed controls

a potent inducer of nodular cirrhosis (16). On the otherhand, among several C-monomethyl derivatives of DAB,the 3'-methyl derivative was considered to be a stronghepatocarcinogen (31), and it also induced marked cirrhotic changes in the liver during carcinogenesis (48). Itseemed appropriate, therefore, to compare the effect ofthioacetamide and 3'-Me-DAB on hepatic AMP deaminaseduring carcinogenesis. Chart 2 depicts this comparison.Here, data were calculated as percent of activity in controlrats fed the basal diet. Throughout the 1st 8 weeks, increases in AMP deaminase activity caused by 3'-Me-DAB

6NUMBEROF INJECTIONS

CHART 3.â€”Increased rat liven adenylic acid deaminase activity

following intra-abdoniinal injections of thioacetamide. Animalsreceived thioacetamide (50mg/kg body weight) on saline injections once daily. AMP deaminase activity was determined 24hr later. Each value represents assays on at least 6 animals,but not more than 12. The vertical lines represent the S.E.

feeding were less than those caused by thioacetamide;however, by the 12th week the magnitude of both wasessentially the same.

Feeding thioacetamide to rats caused, in hepatic parenchymal cells, marked enlargement of nuclei and nucleoli(35) and increases in the amount of nuclear RNA andprotein (26). Adams and Busch (1) reported that i.p.injection of thioacetamide at 50 mg/kg body weight oncedaily for 9 days also produced significant increases innuclear RNA. When we followed their injection regimen(1) and determined the AMP deaminase activity in liver,results as shown in Chart 3 were observed. After 3 injections the AMP deaminase activity was significantlyincreased and these increases were progressive through 6injections.

Since a major feature of hepatic cirrhosis is proliferationof biliary epithelial cells, the elevations in enzyme activityshown in Charts 1 and 2 could be attributed to proliferation of these cells. In previous work, however, evidencewas presented which indicated that the rise in AMPdeaminase activity following feeding of 3'-Me-DAB ora-naphthyl isothiocyanate was not correlated with theproliferation of biliary epithelial cells (23). Although theacute response of the liver to thioacetamide involved majoralterations within parenchymal cells (16, 18, 19, 24, 26,35, 45), and with massive doses, centrilobular necrosis(4), bile duct proliferation was apparently delayed for 2â€”3weeks (19). In view of this, it seems unlikely that therise in enzyme activity shown in Chart 3 was associatedwith a pronounced shift in the cellularity of liver. It wasinteresting to note Opie's observation that the incidenceof liver tumors induced with DAB was independent of thedegree of cirrhosis (33), and Cantarow subsequentlyreached a similar conclusion concerning 3'-Me-DABhepatocarcinogenesis (10).

Weeks on Diets



TREATMENT AND NO.OP INJECTIONSRNA

CONTZNT.

Nuclear fraction(pg/mg nuclear protein).

Cytoplasmic sap fraction(pg/mg sapprotein)Saline0(15)37.5Â±3.1(14)23.0Â±0.91(9)

35.7Â± 1.3(6) 20.9Â±0.82(12)37.4Â±0.1â€”3(12)40.8Â± 3.4(12) 25.8Â±0.84(11)41.6Â±2.9â€”6(9)36.1 Â± 1.5(9) 22.3Â±0.59(12)38.1Â± 1.9(12) 21.9Â±0.6Thioacetamide1(9)

29.9Â± 1.5(6) 22.2Â±0.72(12)33.6Â±2.0â€”3(12)46.9Â± 2.6(12) 33.6Â±3.86(9)46.4Â± 0.2(9) 22.5 Â±0.69(12)54.4Â±3.1(12)23.3Â±1.3


When relatively low concentrations of thioacetamidewere injected daily, the RNA content of the nuclei increased (26). Chronic feeding also caused increases innuclear RNA, but no increases in the RNA content ofcytoplasmic sap were observed (18). When rats weregiven higher, multiple injections of thioacetamide and theRNA of the cytoplasmic sap was separated into 2 fractionsby a modification (40, 50) of Kirby's phenol extractionmethod (21), the amount of RNA in the fraction whichwas released by phenol into the aqueous phase increasedmarkedly (1). Since the rise in enzyme activity wasapparent after the 2nd injection, it was important to determine whether increases in nuclear and cytopla.smic sapRNA also occurred this soon. These 2 cellular fractionswere separated by centrifugation from livers homogenizedin 0.25 M sucrose and the RNA concentration was determined by the Schmidt-Thannhauser-Schneider procedure(47). The data are shown in Table 1. After 3 injectionsof thioacetamide, nuclear RNA was markedly increased.After 9 injections, RNA accumulations in the nucleuswere even more pronounced. The total RNA content ofthe cytoplasmic sap fraction was relatively unchanged;however, a slight transient rise was usually observed after3 injections.

The increase in the size of hepatic nuclei and nucleolifollowing thioacetamide administration has been welldocumented (16, 18, 19, 24, 35, 45); however, in none ofthese experiments was a time course study made followingmultiple injections of relatively high concentrations.Adams and Busch (1) suggested that thioacetamide inter

TABLE 1

THE RIBONUCLEIC ACID (RNA) CONTENT OF NUCLEAR AND CYTO

PLASMIC SAP FRACTIONS IN THE LIVERS OF RATS RECEIVING

INTRAABDOMINAL INJzcTION5 OF THIOACETAMIDEO

NUCLEOLINUCLEI DIAMETERS

T T.1

T#â€˜@â€˜THIOACETAMIDE

THIOACETAMIDE

II

9C,)z0a:U

U)z0a:C)

7

VSALINE

/01 3 6 9Numberof Injections

4

3

2@i@ SALINE

01. . I/01 3 6 9

Number of Injections

CHART 4.â€”The effect of thioacetamide injections on the diameten of hepatic nuclei and nucleoli. Each value represents theaverage of measurements on 2 to 4 individual rat livers . Liverswere fixed in 10% fonmalin-1% calcium chloride (5) and imbeddedin paraffin. Sections were cut at 5 p and stained with Schiffmethylene blue (42). In each liver at least 100 nuclei and thecontained nucleoli were measured. Vertical lines represent S.E.

fered with the release of nucleolar RNA into the cytoplasmwithout appreciably inhibiting synthesis. Since we hadobserved increases in nuclear RNA after 3 injections ofthioacetamide, the diameters of nuclei and nucleoli infixed stained sections were measured after animals hadreceived 1, 3, 6, or 9 injections of thioacetamide. Thedata are shown in Chart 4. The diameters of nuclei andnucleoli increased throughout the 9 injections; however,with the nuclei most of the increase occurred during the1st 6 injections.

It was important, next, to determine whether any relationships existed between increases in enzyme activity,nuclear RNA, and the size of nuclei and nucleoli. These4 parameters of hepatic response to thioacetamide injections were calculated as percent of the same parameters insaline injected animals. The percentage values obtainedare depicted in Chart 5. This expression of the data mdicated reasonably close correlations between increases innuclear RNA and nuclei diameters, whereas increases inenzyme activity and nucleoli diameters appeared to bemore closely correlated. In none of these cases was thereevidence to support a conclusion that increases in onemember of a correlated pair necessarily gave rise to increases in the 2nd member.

Busch et al. (8) suggested that the nucleus was the majorsite of RNA biosynthesis, and later, that within thenucleus the nucleolus was probably a major site of RNAbiosynthesis (9). Among the 3 major categories of cellularRNA, viz., messenger RNA (mRNA), ribosomal RNA(rRNA), and transfer RNA (tRNA) (38), informationgathered from a variety of cells indicated an associationbetween the nucleolus and the synthesis of both rRNA(6, 14, 15, 28, 41) and mRNA (9, 39). Studies with ratliver nuclei indicated the presence of both ribosomal-like

a Livers were homogenized in 0.25 M sucrose and fractionated

by centnifugation (37). RNA was separated by the SchmidtThannhauser-Schneider procedure and determined colonimetnically by reaction with orcinol (47). Animals received thioacetamide (50 mg/kg body weight) or saline injections once daily andwere killed 24 hr after the last injection. Numbers in parenthesesindicate numbers of animals. Values reported are averages withS.E.



3 5 7 9 /01Number of Injections

3 5 7 9


Non-NucleolarNucleorRNA Nucleolar RNACl)a,

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Number of Injections6 9

CHART 5.â€”Comparison of the effect of thioacetamide on nucleidiameters and nuclear RNA and on nucleoli diameters and AMPdeaminase activity. Values plotted were calculated:

value in livers of animals injected with thioacetamidevalue in livers of animals injected with saline X

RNA (43) and messenger-like RNA (20, 43). Increasesin the amount of hepatic nuclear RNA following thioacetamide administration have been well documented(1, 18, 26); however, thioacetamide appeared to exert noappreciably influence on the rate of hepatic nuclear RNAbiosynthesis (1, 18). In 1 of these studies (1) RNAsynthesis was determined by the rate at which orotic acid2-'4C was incorporated into subcellular fractions of theliver. Here, thioacetamide suppressed nuclear RNA biosynthesis, enhanced microsomal RNA biosynthesis, buthad no appreciable effect on nucleolar RNA biosynthesis(1). Since these authors considered that pool sizes mayhave influenced their rate studies (1), it seemed advisableto reinvestigate the influence of thioacetamide on theincorporation of orotic acid-6-'4C at several intervalsduring a 9-day injection regimen and to select methodsfor the subfractionation of nuclear RNA which wouldpermit some separation of nucleolar RNA from the rest ofthe nuclear RNA.

In 2 separate cytologic studies, it was demonstrated (a)that extraction with strong NaC1 solutions at neutralityremoved DNA and left the nucleolus with much of itsribonucleoproteins intact (32), and (b) with the furtheraid of autoradiography that nucleolar RNA was heavilylabeled with PH prior to appreciable labeling of eitherchromatin or cytoplasmic RNA (29). Allfrey and Mirsky(2) believed that the RNA in a nuclear residue which hadbeen successively extracted with pH 7.1 phosphate bufferand 1 M NaCl included nucleolar RNA, and isotopicstudies with orotic acid-2-'4C and adenosine-8-'4C demonstrated that this fraction incorporated isotope at greaterrates than the rest of the nuclear RNA. Busch et al. (7)believed that this method (2) as well as the modified phenolextraction methods of Yamana and Sibatani (40, 50) hadinadequacies when judged by cytologic criteria; nevertheless, it seemed to us that the neutral buffer NaC1 extraction procedure could be expected to yield 2 subfractions ofhepatic nuclear RNA showing markedly different labelingrates.

When rat liver nuclei were purified by centrifugation

CHART 6.â€”The effect of thioacetamide on the amount of RNAin NNN and â€œnucleolanâ€•subfractions of rat liver nuclei.

Nuclei were separated from liver homogenates (37) and thenpurified by centnifugation in 2.2 M sucrose (13). Nuclear RNAwas separated into â€œnon-nucleolarâ€•nuclear RNA (NNN) andâ€œnucleolarâ€•RNA subfractions by the method of Allfney andMinsky (2) . Ribonucleoproteins in each subfnaction were washedas described by Steele et al. (44). The quantity of RNA was detenmined by reaction with oncinol (47), the quantity of proteinby the method of Lowry et al. (27) . Each value represents averages determined for 11 to 16 animals. Calculation of t valuesshowed the following averages to be different at a 0.05 probabilitylevel:A. NNN RNA

1. Both 3 and 6 saline injections exceeded corresponding thioacetamide injections.

2. 9 thioacetamide injections exceeded 9 saline injections.3. Both 3 or 6 saline injections exceeded 9 saline injections.

B. â€œNucleolanâ€•RNA1. Both 3 and 9 thioacetamide injections exceeded correspond

ing saline injections.2. Both 3 and 9 saline injections exceeded 6 saline injections.3. Both 3 and 9 thioacetamide injections exceeded 6 thioaceta

mide injections.

in sucrose (13) and the ribonucleoproteins subfractionatedinto NNN and â€œnucleolarâ€•fractions (2) data as shown inChart 6 were obtained. After 3 or 6 injections, the NNNRNA concentration of the thioacetamide treated animalswas less than that of saline controls. After 9 thioacetamide injections the NNN RNA concentration approachedprevious saline control levels, but animals given 9 salineinjections sustained significant losses of NNN RNA. Wehave no explanation for this latter development, but itwould not appear to influence greatly the question ofthioacetamide's effect on liver NNN RNA. The â€œnucleolarâ€• RNA concentration after 3 or 9 thioacetamideinjections was significantly higher than the concentrationin saline controls. After 6 injections both saline andthioacetamide animals showed significant losses of â€œnucleolarâ€•RNA. We have no explanation for this biphasicchange in the concentration of RNA, but since it occurredin both treatment groups, again it would not appear to begermane to the question of thioacetamide's effects onâ€œnucleolarâ€•RNA. These data suggested that the increase in hepatic nuclear RNA shown in Table 1 andChart 5 and reported previously by others (1, 18, 26)primarily involved the accumulation of â€œnucleolarâ€•RNA.



TREATMENT AND NO.OP INJECTIONSINCORPORATION

INTO â€œNON-NUcLEOLARâ€• Nucu@ziRA.

. .30-Mm incorporation.

.60-Mmincorporationcpnh/mg

Proteinrpm/mg RNAcpm/mg Proteincpm/rngRNASaline3(6)

110 Â± 10(6) 1,960Â± 280(5) 120 Â± 30(5) 1,880Â±1906(6)120 Â± 20(6) 2,160Â± 650(8) 140 Â± 20(8) 2,350Â±3909(8)60 Â± 10(8) 1,340Â± 140(8) 90 Â± 20(8) 2,100Â±470Thioacetamide3(5)

200 Â± 20(5) 4,560 Â± 550(6) 220 Â± 20(6) 5,020 Â±8606(6)170 Â± 20(6) 3,740 Â± 480(7) 210 Â± 30(7) 4,730 Â±8509

Saline(8)

120 Â± 10(7) 2,530Â± 1,000(8) 110 Â± 10(8) 2,135 Â±380INCORPORATION

INTO â€œNuci@Eoz.*zâ€•iRA3(6)

540 Â± 80(6) 14,050 Â± 1,850(6) 520 Â± 80(6) 13,520 Â±1,7006(6)490Â±40(6)15,120Â±1,120(8)680Â±130(8)23,430Â±1,2809(8)

710 Â± 100(8) 14,470Â± 1,720(8) 690 Â± 110(8) 16,420Â±2,260Thioacetamide3(6)

760 Â± 90(6) 12,140Â± 1,160(6) 970 Â± 90(6) 16,680Â±6406(6)360 Â± 50(6) 10,250Â± 750(7) 740 Â± 150(7) 19,610Â±2,0809(6)400 Â± 80(8) 6,180Â± 930(8) 470 Â± 40(8) 8,330Â± 1,021


a Animals were injected intnaabdominally once daily with either saline or thioacetamide (50 mg/kgbody weight). Animals were injected intnaabdominally with 3.3 @cof orotic acid-6-14Cin 2 ml saline24 hr after the last dose. Either 30 or 60 mm later animals were killed by cervical fracture. Livers

were perfused with chilled 0.9% saline and then with 0.25 Msucrose.Livers were homogenized in 0.25 M sucrose and nuclei separated by centnifugation at 1250 X g (36)

and purified by the method of Chauveau (13). RNA was separated into â€œnon-nucleolarâ€•nuclear(NNN) and â€œnucleolarâ€•subfractions by the extraction procedure of Allfrey and Mirsky (2). Ribonucleoproteins in each subfraction were precipitated with 0.75 N perchionic acid. The precipitateswere washed twice with 0.75 Nperchloric acid and twice with 90% ethanol containing 2% sodium acetate(44). Ribonucleoproteins were solubilized in 1 N NaOH and aliquots were withdrawn for determination

of isotope concentration, RNA concentration, and protein concentration. Isotope concentration wasdetermined in a gas flow Geiger counter fitted with a â€œMicromilâ€•window. Counts were corrected forbackground and self-absorption. RNA concentration was determined by the orcinol method (49)using yeast RNA as a standard. Protein concentration in subfractions was determined by the methodof Lowry et al. (27) using crystallized bovine albumin as a standard. Numbers in parentheses indicatenumbers of animals. Values reported are averages with S.E. Calculation of t values revealed thefollowing treatment averages to be different at a probability level of 0.1 or better:

A. NNN RNA (cpm/mg protein)1. 30-mm incorporation; 3 and 6 thioacetamide injections exceeded corresponding saline controls.2. 60-mm incorporation; 3 and 6 thioacetamide injections exceeded corresponding saline controls.

B. NNN RNA (cpm/mg RNA)1. 30-mm incorporation; 3, 6, and 9 thioacetamide injections exceeded saline controls.2. 60-mm incorporation; 3 and 6 thioacetamide injections exceeded saline controls.

C. â€œNucleolarâ€•RNA (cpm/mg RNA)1. 30-mm incorporation

a. 3 thioacetamide injections exceeded saline controls.b. 6 and 9 saline controls exceeded 6 and 9 thioacetamide injections.

2. 60-mm incorporationa. 3 thioacetainide injections exceeded saline controls.b. 9 saline controls exceeded 9 thioacetamide injections.

D. â€œNucleolarâ€•RNA (cpm/mg RNA)1. 30-mm incorporation; 6 and 9 saline controls exceeded 6 and 9 thioacetamide injections.2. 60-mm incorporation; 9 saline controls exceeded 9 thioacetamide injections.

Adams and Busch (1) reported increases in both phenol (50) to be similar to the â€œnucleolarâ€•fraction obtained byextractable RNA (p-RNA) and residual RNA (r-RNA) the method of Alifrey and MirSky (2). Since the heterofollowing 9 thioacetamide injections; only 1 of these sub- geneity of nucleolar RNA has only been realized and notfractions, r-RNA, was considered by Yamana and Sibatani fully characterized (7), it seems as reasonable to attribute

TABLE 2THE EFFECT OF THIOACETAMIDE ON THE RATE OF ORo@nic AciD-6-â€•C INCORPORATION

INTO 2 SUBFRACTIONS OF NUCLEAR RIBONUCLEIC ACID (RNA)a




differences in our data and that of Adams and Busch (1)to differences in the partition of the various RNA speciesamong the fractions obtained by the 2 extraction methods,i.e., the Yamana-Sibatani method (40, 50) and the AllfreyMirsky method (2), as it does to attribute it to differencesin the response of the animals to thioacetamide.

The effect of thioacetamide upon the incorporation oforotic acid-6-'4C into NNN and â€œnucleolarâ€•RNA aftera 30- or 60-miii in vivo incorporation period is shown inTable 2. Since thioacetamide caused increases in nuclearRNA (see Chart 6) and nuclear protein (18, 26), specificactivities were calculated 2 ways, viz., (a) on the basis ofprotein content, and (b) on the basis of RNA content.From the data, and the statistical analysis of the data, itwas clear that thioacetamide, whether injected for 3, 6,or 9 days, caused increased synthesis of NNN RNA. Itwas equally clear that 3 injections of thioacetamide significantly increased the synthesis of â€œnucleolarâ€•RNA.After 6 or 9 injections of thioacetamide, however, thespecific activities of â€œnucleolarâ€•RNA, irrespective ofmethod of calculation, were lower than the specific activities of corresponding saline controls. Whether thislatter change in specific activity actually reflects a diminished synthesis of â€œnucleolarâ€•RNA or a decrease in specificactivity due to increases in protein and RNA content ofthe nucleolar fraction cannot be determined finally withthe present data. Recently, 2 separate studies were published in which thioacetamide caused increased â€œnucleolarâ€•RNA synthesis. Koulish and Kleinfeld (25) fed thioacetamide to rats for 2 weeks and then determined autoradiographically the rate at which tritiated cytidinelabeled liver parenchymal cells. Both â€œnucleolarâ€•andNNN RNA synthesis was increased (25). Tsukada andLieberman (46) injected orotic acid-6-'4C into rats andisolated nucleoli from rat liver nuclei suspensions. Thioacetamide injections enhanced the rate of precursor incorporation into â€œnucleolarâ€•RNA (46). Koulish andKleinfeld (25) suggested that the data of Adams andBusch (1) may have been influenced by the short incorporation intervals. Nevertheless, neither our data northat of others (25, 46) supports the conclusion of Adamsand Busch (1) that thioacetamide caused no increase inrat liver nuclear RNA synthesis.

Since under their conditions Adams and Busch (1) observed no increase in nuclear RNA biosynthesis, they suggested that the accumulation of nuclear RNA was associated with some effect of thioacetamide on the release ofnucleolar ribonucleoproteins into the cytoplasm. Thedemonstration that thioacetamide increased nuclear RNAsynthesis in the present study and in other studies (25, 46)suggests the possibility that nuclear RNA might be expected to accumulate if synthesis were increased without aconcomitant increase in the rate at which nucleolar RNAwas released.

A previous study (23) showed that 3'-Me-DAB causedincreases in AMP deaminase activity of the precancerousliver and that these increases fit criteria defined for a keychange. The data presented in this report establishedthat a second kind of hepatocarcinogen, thioacetamide,also caused increases in the AMP deaminase activity ofprecancerous rat liver despite marked differences in chemi

cal structure and characteristics. Increases in enzymeactivity were elicited by either chronic or acute modesof thioacetamide administration, and were accompaniedby certain morphologic and metabolic changes amonghepatic cells. At this juncture it is not known whetherany of these changes are interrelated or whether they arerelated with sequential events leading to malignant transformations in the liver. This latter choice can be madeonly in systems in which it is possible to prevent or delayhepatocarcinogenesis (36). Since significant increases inAMP deaminase activity occurred within 3 days following thioacetamide injections, underlying mechanisms cannow be studied, perhaps in a manner analogous to thestudies of Weber and Singhal (49) on cortisone-inducedsynthesis of hepatic enzymes associated with carbohydratemetabolism.

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1965;25:596-603. Cancer Res Donald E. Kizer, B. C. Shirley, Bettye Cox, et al. and Nuclear Ribonucleic Acid Metabolism in Rat LiverEffect of Thioacetamide on Adenylic Acid Deaminase Activity

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