[CANCER RESEARCH 37. 2226-2231. July 1977]

Effects of Dietary Feeding of Aflatoxin B, on Ribosomal RNAMetabolism in Rat Liver1

Steven J. Smith,2 Keith C. Deen, William J. Calhoun, and Howard F. Beittenmiller

Department of Pharmacology, Pennsylvania State University, The MîltonS. Hershey Medical Center, Hershey, Pennsylvania 17033

SUMMARY

After 1 week of dietary feeding of Aflatoxin B, (AFB,) (1 or10 ppm) to male Fischer rats, sucrose gradient analysisrevealed decreases in the incorporation of [3H]orotic acid in

vivo into 45 S hepatic nRNA and decreased concentration(A2W1)of 28 S nRNA when compared with controls fed anidentical diet minus AFB,. Sucrose gradient analysis of hepatic microsomal rRNA showed decreases of a similar magnitude in labeling in vivo and in A2til,of 28 S rRNA. Labelingand A2(ioof the 18 S rRNA were unchanged. Assay of RNApolymerase I activity in isolated hepatic nuclei demonstrated that this enzyme activity was not diminished in ratsfed the AFB, diet from that of the controls. Feeding of AFB,for 1 to 6 weeks resulted in progressive decreases in A2fi„of28 S nRNA and in both label and A2W,in microsomal 28 SrRNA. These effects are time and dose related, since 1 weekof a diet containing 10 ppm produced changes in nuclearand ribosomal 28 S RNA similar to those observed after 4 to6 weeks of a diet containing 1 ppm. Sixteen hr after a singleinjection of AFB, (1 mg/kg i.p.), the same defects in RNAmetabolism occurred as described above for 1 ppm for 4 to6 weeks and 10 ppm for 1 week. In contrast to the chronicfeeding studies, after an acute injection these effects eventually disappear. These data suggest that early progressivelesions in the maturation of hepatic 28 S rRNA are producedduring chronic feeding of a diet containing AFB,. Suchdefects in processing of ribosomal precursor RNA may be acharacteristic feature of chemical hepatocarcinogenesis.

INTRODUCTION

Although extensive biochemical studies have been carried out on the effects of a single injection or p.o. dose ofhigh levels of potent hepatocarcinogens on rat liver RNA (2,9, 12, 25, 32, 34, 42, 50), few studies have been carried outconcerning the effects of chronic feeding of low doses ofthese agents on RNA metabolism (1, 10). AFB,3 is a potenthepatocarcinogen that produces marked alterations in cellular RNA metabolism (2, 10, 25, 32, 50). After a singleparenteral injection, most investigators have reported (2,

1 Supported in part by Grant CA16804 and by a specialized Cancer CenterGrant 1 P30 CA18450-01 awarded by the National Cancer Institute, Department of Health. Education and Welfare.

2 To whom requests for reprints should be addressed.3 The abbreviations used are: AFB,. aflatoxin B,; TCA, trichloroacetic acid;

hnRNA, heterogenous nuclear RNA.Received January 6, 1977; accepted April 19, 1977.

25, 32) that this agent has little effect on the synthesis ofribosomal precursor RNA (45 S RNA) but significantly inhibits synthesis of mRNA precursors. Such differential effects on synthesis of 45 S rRNA and mRNA precursors maybe characteristic of chemical hepatocarcinogens (2, 9, 12,25, 32, 50).

In recent years increasing attention has been given to theintranuclear posttranscriptional processing of mRNA andrRNA precursors (28, 38). The exact mechanism(s) of processing and transport of mRNA precursors has not beenelucidated. With regard to rRNA, the processing involvesthe cleavage at specific sites of a large precursor moleculeof 45 S rRNA to 18 and 28 S rRNA, which appear in thecytoplasm as free 60 and 40 S subunits (28).

As part of their mechanism of action, various agents canaffect processing of 45 S rRNA and either increase or decrease the concentration of newly formed ribosomal sub-units and rRNA in the cytoplasm (22, 28, 38). Moulé(22)reported that, after a single injection of AFB, to rats, the 60S ribosomal subunit containing 28 S rRNA did not appearin the cytoplasm, whereas the emergence of 40 S subunitcontaining 18 S rRNA was unimpaired.

This study was designed to examine whether chronicfeeding of AFB, to rats affects synthesis and/or processingof ribosomal precursor RNA. Our results indicate that within1 week of feeding a diet containing 1 ppm of AFB,, abberra-tions occur in the maturation of 28 S rRNA that may resultfrom defective intranuclear processing of rRNA precursors.This defect in the processing of 28 S rRNA is time and dosedependent. Under the same conditions of treatment, synthesis of ribosomal precursor RNA appears to be unaffected.

MATERIALS AND METHODS

Animals and Isotopes

Male Fischer rats (Charles River Breeding Laboratories,Wilmington, Mass.) weighing 120 g were fed ad libitum asemisynthetic agar gel diet for 1 week (50). AFB, (Makor,Inc., Jerusalem, Israel) was dissolved in corn oil and mixedin the agar gel diet at concentrations of 1, 5, and 10 ppm. Afew experiments were carried out using dietary concentrations of 20 and 50 ppm. Control rats were fed the identicaldiet minus the carcinogen. For injection, AFB, was dissolved in dimethyl sulfoxide and administered i.p. (1 mg/kg) in a final volume of 0.05 ml. For labeling of nRNA ormicrosomal RNA/n vivo, each rat received i.p. 50 or 100 fj.C\

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Effects of AFB, on Processing of 45 S rRNA

of [5-3H]orotic acid (18 mCi/mmole; New England Nuclear,

Boston, Mass.) and was decapitated 20 min or 2 hr later,respectively.

Isolation of Nuclei

Isolation of Nuclei for In Vivo Labeling Studies. For eachtime point or experimental group, livers from 3 or 4 ratswere pooled. Minced liver tissue was homogenized in 2.4 Msucrose (1:11, w/v) containing 3.3 mM CaCI2 with 4 up-and-down strokes in a Teflon-glass homogenizer (0.025-inch

pestle clearance). After successive filtrations through 2 and4 layers of cheesecloth, the homogenate was centrifuged at40,000 g for 60 min to sediment the nuclei (37).

Isolation of Nuclei for the RNA Polymerase I Assay. Theminced liver tissue was placed in a freshly prepared solutionof 2.3 M sucrose containing 15 mM MgCI2 and 0.25 mwspermine and homogenized as above (37). The homogenatewas centrifuged at 40,000 x g for 50 min to sediment thenuclei. Nuclei were prepared and maintained in hypertonicsucrose before the enzyme assay to prevent the loss ofsoluble polymerases or other endogenous macrospecies(19).

Under the phase-contrast microscope, nuclear prepara

tions obtained by either the sucrose:calcium or sucrose:magnesium procedure were highly purified. No differencewas observed between the purity or gross morphology ofisolated nuclei of carcinogen-treated rats and controls.

Isolation of Microsomes. The cytoplasmic microsomalfraction was prepared in isotonic sucrose as described previously (36).

Acid-soluble Pool. Aliquots of homogenates were used todetermine the specific activity of the total acid-soluble poolafter labeling in vivo with [3H]orotic acid (23).

Extraction and Analysis of RNA

Nuclei or microsomes were homogenized in a solutioncontaining 0.3% sodium dodecyl sulfate, 0.14 M NaCI, and0.05 M sodium acetate (pH 5.1) for 1 min (15 strokes) with aloosely fitting Teflon pestle. After addition of 0.05 M sodiumacetate-saturated phenol containing 0.1% 8-hydroxyquino-

line, the sample was homogenized again for 1 min. Thesuspension was shaken successively at 65°for 10 min and

then for 30 min at room temperature. For microsomal RNAextraction, heating at 65°was omitted (36).

The mixture was centrifuged at 17,000 x g for 10 min, andthe aqueous phase was removed; this was followed by 2reextractions of the aqueous phase with phenol at roomtemperature. The RNA was precipitated overnight at -20°

with 2.5 volumes of ethanol containing 2% potassium acetate (36). The precipitate was dissolved in glass-distilledwater, reprecipitated in ethanol-potassium acetate, and re-

dissolved in water. Absorbance at 260 nm was measured;the radioactivity of the solution was then measured in 10 mlof NEN-949 (New England Nuclear) toluene-based fluor using a Beckman LS-100 liquid scintillation counter.

Sucrose Gradients and Analysis of Specific Activity

Between 0.2 and 1 mg of RNA was layered over 10 to 40%

sucrose gradients (38 ml) containing 0.1 M NaCI, 1.0 mMEDTA, and 0.01 M sodium acetate, pH 5.1 (36). The gradients were centrifuged in a Spinco SW 27 rotor at 26,000rpm for 16 hr at 5°.Analysis of gradients was carried out

with the aid of an ISCO automatic fractionator system.Absorbance at 254 nm was transcribed with a Honeywellrecorder and is described in this paper as A260for differentsedimentation classes of RNA. Radioactivity of each fraction was determined by liquid scintillation (3).

The specific activity of RNA (dpm/mg RNA) was determined by pooling the 3 gradient fractions that constitutedthe peak and by precipitating them in ethanol overnight asdescribed above. The precipitate was dissolved in water,and its absorbance at 260 nm was measured; the radioactivity of the solution was then measured by liquid scintillationas above. For these calculations, 1 mg of RNA was equivalent to 20 A2(lllunits.

RNA Polymerase I Assay

RNA polymerase I activity in isolated hepatic nuclei wasassayed essentially as described by Jacob (15). The incubation was carried out in the presence of a-amanitin. Since a-

amanitin is a specific inhibitor of nucleoplasmic RNA polymerase (Form II), the residual activity of the enzyme assayed in low ionic media in the presence of the toxin represents nucleolar RNA polymerase I activity (15). AlthoughRNA polymerase III activity is also being measured, thisactivity is very low. The assay mixture contained in a finalvolume of 0.4 ml: 42 /¿moles Tris-HCI buffer, pH 8.5; 2

Amóles MgCI2; 2.5 Amóles NaF; 1.1 Amóles dithiothreitol;0.3 Amóles each of ATP, GTP, and CTP; 0.008 /¿molesUTP;0.0013 /¿molesUTP-[14C] (53 mCi/mmole; New England Nuclear; 0.25 /¿ga-amanitin; and nuclei containing 20 to 40 /¿gof DNA. The assay was carried out at 37°for 10 min. Under

these conditions the concentration of UTP or enzyme wasnot rate limiting. A linear increase in enzyme activity occurred using nuclei containing a range of DNA concentrations of 10 to 60 fig.

The reaction was terminated by adding 0.1 mg of UTP ascarrier and 1.5 ml of 10% TCA containing 0.04 M Na4P2O7.Each enzyme assay was carried out in triplicate. Blankswere obtained by adding substrates after precipitation withTCA. The reaction mixtures were filtered through WhatmanGF/C filter paper under light vacuum and washed 4 timeswith 2 to 4 ml of 5% TCA containing 0.02 M Na4P2O7. Thedried filter discs were placed in glass vials, 8 ml of NEN-949

scintillation fluid were added, and the vials were counted ina Beckman LS-100 scintillation counter. Data are expressed

as pmoles of UMP incorporated per mg of DNA.

Determination of DNA

To estimate DNA content in nuclei used for the RNApolymerase I assay, aliquots of isolated nuclei were used.DNA was extracted by acid hydrolysis (23) and determinedusing the procedure of Burton (4).

RESULTS

Effect of AFB, Treatment of Rats on Sucrose GradientPatterns of Hepatic Nuclear and Microsomal RNA. Follow-

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S. J. Smith et al.

ing a single i.p. injection of AFB, (1 mg/kg), Moulé(22)reported the absence of 60 S ribosomal subunits and 28 SrRNA in the cytoplasm. Under the more physiological dietary route, we examined the effects of AFB, on labeling invivo and the concentration (A2(ill)of microsomal 18 and 28 SrRNA.

More degraded RNA was present in nuclear as well asmicrosomal RNA from AFB,-treated rats than from that ofcontrols. These and other low-molecular-weight materialswere removed by reprecipitation in ethanohpotassium acetate for 3 hr at -20°just before preparation of the sucrose

gradients. The only effect on sucrose gradient profiles wasthat much less absorbance was recorded in the 4 to 7 Sregion of sucrose gradients from AFB.-treated rats becauseof the loss of degraded RNA. Thus, the amount of 4 to 7 SRNA from RNA of AFB,-treated rats and from control ratswas roughly equal.

Sucrose gradient analysis of hepatic microsomal rRNAextracted from rats fed AFB, showed a dose- and time-related inhibition of 28 S rRNA labeling and A2t;i,(Chart 1).Sucrose gradient analysis revealed that decreases in labeling in vivo (specific activity) occurred in microsomal 28 SrRNA after only 3 days of feeding a diet containing 1 ppm ofAFB, (data not shown). Gradient patterns obtained fromrats fed dietary levels of 1 ppm for 1 to 2 weeks or 10 ppm for3 days were very similar and showed decreased labeling andA2„„of 28 S rRNA and no effect on 18 S rRNA (Chart 1ß).Chart 1C shows that similar gradient patterns were obtainedfrom microsomal rRNA of rats fed AFB, at 1 ppm for 4 to 6weeks, at 10 ppm for 1 week, or at 16 hr after a single i.p.injection of 1 mg of this toxin per kg. An enhanced inhibitory effect on the labeling and A21il,of 28 S rRNA was observed. Slight increases in labeling and A21ii,of 18 S rRNAwere probably due to breakdown products of the 28 S rRNA(Chart 1C).

In order to examine the possibility that defective maturation of 28 S rRNA was a result of intranuclear aberrations inprocessing of ribosomal precursor RNA, rats were treatedas in Chart 1, and sucrose gradient analysis of nRNA wascarried out. Chart 2 demonstrates a similar dose- and time-related effect as was observed in Chart 1. Dietary feeding ofAFB, at 1 ppm for 1 to 2 weeks or at 10 ppm for 3 daysproduced a decrease in the labeling in vivo and A2I1(,of 45 SnRNA and a decrease in the A28(,of 28 S nRNA (Chart 2B).These effects were increased in rats fed AFB, at 1 ppm for 4to 6 weeks, at 10 ppm for 1 week, or 16 hr after a single i.p.injection of 1 mg of AFB, per kg (Chart 2C). High-molecular-weight nRNA is a mixture of hnRNA (mRNA precursors) andrRNA precursors. Although 45 S nRNA contains little A260and label from hnRNA (after 20 min of labeling), the 28 SRNA may contain significant label from hnRNA (24). In 2experiments sucrose gradient analysis of nucleolar RNAfrom rats fed 10 ppm of AFBi for 1 week demonstratedsimilar effects as shown in Chart 2C.

Specific Activity of Hepatic Nuclear and MicrosomalRNA following AFB, Treatment of Rats. After labeling invivo with [3H]orotic acid for 2 hr, the specific activity of the

total microsomal rRNA or 28 S microsomal rRNA of rats fedAFB, or given an injection of a single dose was not changedfrom that of the controls, because corresponding decreases

4-7S 18S 28S

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FRACTION NUMBERChart 1. Effects of AFB, on sucrose gradient profiles of microsomal rRNA.

The chart is a composite of 2 types of experiments: (a) AFB, (1 mg/kg) wasinjected i.p. into male Fischer rats, and the animals were killed 16 hr later; (b)animals were fed a diet containing AFB, for 1 to 6 weeks at a level of 1 ppm orfor 3 days to 1 week at 10 ppm. Two hr after a single i.p. injection of [3H]oroticacid (100 /iCi/rat), animals were killed at 8:00 am. Microsomal rRNA wasextracted, and sucrose gradient analyses were carried out as described in"Materials and Methods." Three to 4 rats were used for each experiment, and

3 experiments were carried out for each treatment regimen. Control rats werefed identical diets minus the carcinogen. In each experiment, control ratsand experimental animals were killed on the same day. A. sucrose gradientsof microsomal rRNA from control rats; B, effects on sucrose gradient patterns of microsomal rRNA from rats treated with AFB, for 1 to 2 weeks at 1ppm or for 3 days at 10 ppm; C, effects on sucrose gradient patterns ofmicrosomal rRNA from rats treated for 4 to 6 weeks at 1 ppm. for 1 week at 10ppm, or 16 hr after a single i.p. injection of AFB, (1 mg/kg). , A¡™; ,radioactivity.

in labeling and A2(ioof the 28 S rRNA occurred (Chart 1).However, for nRNA, labeling of 45 S RNA decreased morethan the decrease in A2,n,in both rats fed AFB, and inanimals receiving a single injection (Chart 2). In rats fedAFB, at 1 ppm or 10 ppm, decreases in specific activity of 45S RNA were observed (Chart 2). Table 1 shows that 3 and 5days of feeding AFB, at 5 ppm decreased the specific activ-

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Effects of AFB, on Processing of 45 S rRNA

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FRACTION NUMBERChart 2. Effects of AFB, on sucrose gradient profiles of nRNA. The chart is

a composite of 2 types of experiments as described in Chart 1. Animals werekilled 20 min after receiving an i.p. injection of [3H]orotic acid (50 ^Ci/rat).nRNA was extracted, and sucrose gradient analysis was performed as described in "Materials and Methods." The chart shows sucrose gradients of

nRNA extracted from rats treated as described in Chart 1. A, sucrose gradients of nRNA from control rats; B, effects on sucrose gradient patterns ofnRNA from rats fed AFB, for 1 to 2 weeks at 1 ppm or for 3 days at 10 ppm; C,effects on sucrose gradient patterns of nRNA from rats treated for 1 week at10 ppm, or from rats given a single injection of AFB,. , A2to; , radioactivity.

ity of 45 S nRNA. These data are in accord with those ofFloyd ef al. (6), who reported a marked decrease in thespecific activity of nucleolar 45 S nRNA after a single injection of AFB,.

Effects of AFB, Treatment of Rats on RNA Polymerase IActivity. To establish whether the decrease in 45 S rRNAspecific activity was a result of decreased synthesis or increased degradation, we determined the activity of RNApolymerase I in nuclei isolated from rats treated with different regimens of AFB,. In agreement with the data of Saun-

dersef al. (32) and Akinrimisief al. (2) who injected rats withthis toxin and solubilized the polymerases, in rats fed AFBi-

containing diets (1 to 10 ppm) for up to 4 weeks, no decrease occurred in the activity of RNA polymerase I in isolated nuclei (Table 2). At very high dose levels of AFB, (20 or50 ppm), after 1 week of treatment, significant decreases inthis enzyme activity were observed (Table 2). However, in

Table 1Effect of dietary ingestion of AFB, on labeling in vivo of 45 S

rRNA with [3H]orotic acid

Rats were treated with AFB,, nuclei were isolated, and sucrosegradient analyses were performed as described in "Materials andMethods." The peak tubes constituting the 45 S RNA were precipitated, and specific activity was determined as described in "Materials and Methods." The specific activity of 45 S RNA was correctedfor slight changes that occurred in the total acid-soluble radioactivity.

Treatment withAFB,Days1

35ppm5

55Specific

activity of 45 Sdpm/mg)AFB,2067

1278270Control1944

18941876RNA(10-3

xAFB,:

controlratio1.06

0.670.14

Table 2Effect of dietary feeding of AFB, on RNA polymerase I activity in

isolated hepatic nucleiRats were fed diets containing AFB,, nuclei were isolated, and

assay of RNA polymerase I activity was carried out as described in"Materials and Methods." The control values varied slightly on

different days. These values for RNA polymerase I activity in controlrats were 980 ±40 pmoles/mg DNA (n = 30). The data for eachtreatment regimen are expressed as the percentage of the corresponding control value obtained on the same day. The results arethe average of 3 to 5 experiments for each treatment regimen; 3 to 4rats were used for each experiment.

TreatmentTime3

days1week2weeks4weeks5days2weeks1week2weeks1week1

weekDose(ppm)11115510102050Incorporation

ofUMP(pmoles/mg

DNA)1080±80*1070±801440±1301010±90950±1001260±80710±30870±60460±6860± 40%

of corresponding con

trol1201101101201201301201108060Statisticalsig

nificance"NSrNSNSNSNSNSNSNSp

<0.05p< 0.05

" Statistical significance was determined by Student's f test.»Mean ±S.E.c NS, not significant.

rats fed such high levels both body weight and liver weightwere decreased by approximately 30%. Under all other regimens of dietary feeding, no significant decreases in body orliver weight were noted.

DISCUSSION

These data show that an intranuclear defect in processingor maturation of ribosomal precursor RNA occurs within 1week after administration of AFB,. These studies demonstrate that aberrations occur in the sucrose gradient profilesof nRNA and microsomal hepatic rRNA from rats that werefed a diet containing 1 to 10 ppm of AFBt for definedperiods. Diminished labeling in vivo and decreased concentrations of microsomal 28 S rRNA or 45 S nRNA were observed in rats fed AFB,: (a) at 1 ppm for 1 to 6 weeks, (b) at10 ppm for 3 days or 1 week, and (c) 16 hr after a single i.p.

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S. J. Smith et al.

injection of 1 mg/kg. Aberrant maturation of rRNA precursors is dose and time dependent (Charts 1 and 2). Decreased specific activity of 45 S nRNA in AFB,-treated rats(Table 1) was apparently not due to decreased synthesis,since the activity of RNA polymerase I (which synthesizes 45S rRNA) is not decreased under these same conditions oftreatment (Table 2).

Several lines of evidence support the idea that such defects in processing of rRNA precursors occur soon afterexposure to various hepatic carcinogens and may be earlyirreversible lesion(s) related to neoplastic transformation.After injection of various hepatocarcinogens such as ethio-nine (46), AFB, (22), or thioacetamide (43, 44), defects inmaturation of rRNA occur. Swann ef al. (46) observed, similar to our results, that ethionine treatment of rats produceddecreases in processing of hepatic ribosomal precursorRNA that were not due to changes in synthesis of 45 S rRNA.Hepatomas possess much less rough endoplasmic reticu-lum and ribosomes than does normal liver; the degree ofribosome deficiency is greater in more anaplastic tumors (7,13, 21, 24, 27, 29, 45).

Preliminary data from our laboratory4 show that adminis

tration of 40 ppm of diethylnitrosamine via drinking waterfor 1 week produces similar effects on rRNA metabolism asdoes dietary administration of 1 ppm of AFB, for 1 week.Labeling and A2Soof 45 S nRNA and microsomal 28 S RNAare decreased and RNA polymerase I activity is unaffectedby diethylnitrosamine.

In assessing the results of experiments where animalsreceive a single high dose of a particular hepatocarcinogencompared with chronic dietary or p.o. ingestion studies,several differences must be noted. After a single injection ofa hepatocarcinogen, most biochemical alterations, if notall, produced are reversible in surviving animals. For example, inhibjtion of Form II RNA polymerase activity does notpersist after a single treatment with AFB, (2, 32, 50) andprobably most other carcinogens. Furthermore, direct cyto-toxic effects unrelated to carcinogenesis may be produced(5, 27, 31, 45), and a relatively low incidence of hepatomasis produced after a single injection (20, 50).

In contrast, following chronic exposure to low doses ofhepatocarcinogens in the diet or via p.o. ingestion in drinking water, the cumulative effects of these agents after varying periods of time can be measured. Under defined conditions a 95 to 100% incidence of hepatomas occurs within 20to 80 weeks (47-50). After chronic treatment various biochemical changes or lesions can be measured that may notbe reversible with continued dosing. In this regard, after 3to 6 weeks of dietary administration of potent hepatocarcinogens and return to a carcinogen-free diet, there is a lowincidence of hepatomas. After 6 to 8 weeks or more ofdietary feeding of these agents, a relatively high incidenceof hepatomas occurs (17, 18, 26, 30, 51).

With regard to the effects of various chemical hepatocarcinogens on transcription of ribosomal and mRNA, mostinvestigators (2, 9, 10, 12, 25, 32) report decreased RNA

•S. J. Smith, K. C. Deen, L. S. DeFord, and H. F. Beittenmiller. Effects ofIngestion of Diethylnitrosamine on Ribosomal RNA Metabolism in Rat Liver,manuscript in preparation.

polymerase II activity in isolated nuclei or in a solubilizedpreparation after acute (a single injection) or chronic dietary treatment with a chemical hepatocarcinogen. In contrast, RNA polymerase I activity measured under these sameconditions appears to be much less affected, or unaffected,by similar treatment with hepatocarcinogens (1, 2, 9,10,12,25, 32).

Various investigators (6, 16, 43, 44, 46) reported thattreatment with a variety of structurally unrelated chemicalhepatocarcinogens produces decreases in the synthesis of45 S rRNA. In contrast, our data indicate that decreasedspecific activity of 45 S rRNA in AFB,-treated rats appears toreflect increased degradation of newly synthesized RNA andnot decreased synthesis of this RNA.

The possible effects of chemical carcinogens on the template function of DNA or chromatin are controversial. Manyconflicting results (9, 12, 52) concerning template functionand/or the activity of RNA polymerases after treatment withchemical carcinogens may be due to differential loss ofsoluble enzymes and other macrospecies in isotonic mediaduring the preparation of subcellular fractions (19).

Other experiments are in progress in our laboratory relating to the effects of 3'-methyl-4-dimethylaminoazobenzene

on these same parameters of rRNA metabolism. If chronicp.o. ingestion of low doses of structurally unrelated hepatocarcinogens such as AFB,, diethylnitrosamine, and 3'-methyl-4-dimethylaminoazobenzene produce similar defects in processing of ribosomal precursor RNA, the likelihood that these effects are related to hepatocarcinogene-sis would be enhanced. Experiments are in progress on theeffects of these agents on sucrose gradient patterns ofnucleolar RNA and on the separation of hepatic nRNA intomRNA precursor and ribosomal precursor using polyuridy-late or oligodeoxythymidylate-cellulose columns.

With regard to mRNA, decreased (42) or increased (8, 33-35, 40, 41) nucleocytoplasmic transport of some specificspecies of mRNA may occur. Disruption of nucleolar ultra-structure produced by these agents (27, 45) may result indefects in nucleolar-mediated transport of mRNA (11, 14,39, 42) that could be related to carcinogenesis.

The mechanism(s) of the defect in processing of ribosomal precursor RNA is unclear but may be related to aberrations in methylation of 45 S rRNA, or synthesis, transport,and function of ribosomal proteins, or specific nuclear nu-cleases. Future studies must be carried out to elucidate thenature of this lesion and its relevance to carcinogenesis.

ACKNOWLEDGMENTS

We wish to thank Linda S. Deford and Thomas B. Leonard for technicalassistance and Dr. Samson T. Jacob for helpful discussions.

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1977;37:2226-2231. Cancer Res   Steven J. Smith, Keith C. Deen, William J. Calhoun, et al.   Metabolism in Rat Liver

on Ribosomal RNA1Effects of Dietary Feeding of Aflatoxin B

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