Effects of aflatoxin B1 and other carcinogens upon nucleolar RNA of various tissues in the rat

Q 1968 by Academic Press Inc. Experimental

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Cell Research 51, 423-438 (1968)

EFFECTS OF AFLATOXIN CARCINOGENS

UPON NUCLEOLAR RNA OF VARIOUS TISSUES IN THE RAT1 L. R. FLOYD,

Department

B, AND OTHER

T. UNUMA

and H. BUSCH

of Pharmacology, Tumor By-products Laboratory, Baylor College of Medicine, Houston, Tex. 77025, USA Received

September

University,

28, 1967

AFLATOXIN B, is one of the most recently discovered hepatocarcinogens. In 1961 Lancaster et al. [15] found that aflatoxin containing peanut meal, fed to rats for 6 months, causes liver tumors. In 1965 it was reported [l ] that short-term treatment with aflatoxin B, caused the nucleoli of normal rat liver to decrease in size and undergo segregation of fihrillar and granular elements. The present studies were carried out to determine some of the characteristics of the RNA produced by these nucleoli. 4-Nitroquinoline-N-oxide (4-NQO) produces tumors in the skin of mice [18, 191 and transforms hamster embryonic cells into tumor cells [13]. Effects on nucleoli of Ehrlich ascites cells, similar to those seen with aflatoxin B,, have been reported with 4-NQO [16]. These carcinogens and 3’-methyl-4-dimethylaminoazobenzene (3’-MeDAB) have been reported to inhibit the synthesis of RNA in various tissues [7, 10, 14, 251. Since these compounds have such a marked effect on the morphology of the nucleolus, the present studies were designed to determine whether changes occurred in RNA isolated from nucleoli of treated tissues. The RNA was fractionated into various sedimentation classes by centrifugation in linear gradients of sucrose. In all cases, the synthesis of RNA with a sedimentation coefficient at 45s and above was inhibited by these carcinogens. Actinomycin D causes segregation of fibrillar and granular elements in nucleoli and changes the base composition of nucleolar RNA to one which is lower in guanylic and cytidylic acids and higher in adenylic and uridylic acids [21]. It also markedly inhibits the synthesis of RNA in the nucleolus [al], primarily that with a sedimentation coefficient of 45s. In the present study aflatoxin B,, 4-NQO and 3-MeDAB were found to produce similar morpho1 These studies were supported in part by grants from the American Coffin Childs Fund and US Public Health Service CA-08182.

Cancer Society, the Jane

Experimental

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T. Clnuma

and H. Busch

logical effects on the nucleolar elements. However, no changes were found in base compositions at doses sufficient to effect marked inhibition of synthesis of 45s RNA. Simard and Bernhard [23] and Svoboda and associates [29] have noted that many compounds produce segregation of nucleolar elements. From the results of the present studies it would seem that in general substances that block synthesis of nucleolar 45s RNA produce segregation of nucleolar elements. ?c/loreover, the carcinogens, unlike actinomycin D, produce a general inhibition rather than a selective inhibition of nucleolar biosynthetic reactions. MATERIALS

AND

METHODS

Tissues.-Since it was found that tissues varied in their sensitivity to the biochemical and morphological effects of the carcinogens, the tissues with the greater sensitivity were used for studying the effects of each carcinogen. Aflatoxin B1 is a very potent hepatocarcinogen and its effects on rat liver were readily demonstrable in these studies. No reports of hepatocarcinogenic activity have as yet been made for 4-NQO and no success has been achieved in obtaining an inhibition of 45s RNA synthesis in the nucleoli of rat liver with 4-NQO. On the other hand Novikoff ascites cells were found to be inhibited by this carcinogen. Alth.ough 3’-MeDAB is a well known hepatocarcinogen, regenerating rat liver was used instead of normal rat liver because studies have shown that increased cellular proliferation increases susceptibility to its carcinogenic effects [4, 51. Animals-The animals used in these studies were male albino rats, weighing 200-250 g, obtained from the Cheek-Jones Company (Tomball, Texas), and fed ad libitum on Purina Laboratory Chow. Partial hepatectomy was effected by excision of three lobes of the liver so that 30-40 per cent of the liver remained intact. The regeneration was allowed to proceed for 18 h before the animals were sacrified. These animals were anesthetized with diethyl ether and exsanguinated by aortic transection. Before they were excised, the normal livers were perfused with 20 ml of ice-cold isotonic saline solution and then with 20 ml of 0.25 M sucrose containing 3.3 mM CaCl,. For regenerating liver, 10 ml of each of the above solutions were used to perfuse each liver. The excised livers were placed in ice-cold 0.25 M sucrose containing 3.3 mM CaClz and transferred to the cold room (3°C). The tumor used was the Novikoff ascites hepatoma, which was transplanted 6-8 days prior to the experiment. The animals were killed by decapitation and the ascites fluid was drained through an incision in the abdominal wall. There was about 50 ml of this fluid per rat. The ascites fluid was collected in a beaker positioned in an ice slush at 0°C. Isotopes.-2-W-erotic acid, used in these experiments, was obtained from Calbiochem. Its specific activity was 25 &mmole. It was injected via the jugular vein, 3 PC/rat, 10 min before the animals were sacrified. S2P-orthophosphate used in these experiments was carrier-free orthophosphate (Code P-l, Union Carbide Nuclear Co., Oak Ridge, Tenn.). In the experiments on regenerating liver, 10 rats were used Experimental

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RNA

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and 5 rats were injected with 1.5 mc S2P-orthophosphate intravenously via the jugular vein. Ten min later the animals were anesthetized and sacrificed. In the experiments on normal liver and Novikoff hepatoma, each rat was injected with 2 mc of 32P-orthophosphate and killed 30 min later. Carcinogens.-Aflatoxin B1 was supplied as a gift from Dr G. N. Wogan of the Department of Nutrition and Food Science, Massachusetts Institute of Technology. For the studies on acute toxicity the compound was injected intraperitoneally in doses of 50 to 500 pug/kg body weight for 30 min treatment periods. The concentration was 0.5 mg of aflatoxin B1 in 1 ml of dimethylsulfoxide. 3’-Methyl-4-dimethylaminobenzene was synthesized by the method of Giese, Miller and Miller [Ill; 100 mg/kg was injected, i.p.l in corn oil 18 h before the animals were killed. 4-Nitroquinoline-N-oxide was a gift from Dr Hideyo Endo of the Japanese Foundation for Cancer Research. It was injected in doses of 0.5, 2, 5 and 10 mg/rat intraperitoneally in a dimethylformamide solution containing 10 mg/l ml of solution for doses above 0.5 mg and 20 mg/l ml for the 0.5 mg dose. The experiments were terminated 1 h after the injection of the 4-NQO. Isolation of nucleoli.-Both regenerating and normal livers were minced with scissors. The minced tissues were homogenized in 2.3 M sucrose containing 3.3 mM CaCl, (1: 12 w/v) with a Teflon-glass homogenizer, filtered through 4 layers of cheesecloth and centrifuged at 40,000 g for 1 h [26, 281. The 40,000 g nuclear pellet formed was resuspended in 0.25 M sucrose solution (nuclei from 1 g of wet tissue to 1 ml of solution). For release of the nucleoli from the nuclear preparations a Branson sonifier was used, which had a power of 75 watts. The nuclear suspension, 50 ml, was sonicated for about 30 set in a rosette held in an ice slush at 0°C. The suspension was sonicated until no nuclei were observed under the light microscope. Then, 20 ml portions of the sonicate were each underlayered with 20 ml of 0.88 M sucrose and spun at 2000 g for 20 min for purification of the nucleoli. For isolation of nucleoli from Novikoff ascites cells, the following procedure was used [20]. After collecting the ascites fluid from the rats, the cells were packed by centrifuging them at 4000 g for 20 min with a GSA rotor in a Servall RC-2 centrifuge. The packed cells thus obtained were weighed; 8-10 g of packed cells were usually obtained per rat. These cells were resuspended in 2.0 M sucrose with 3.3 mM CaCl, in a ratio of 12 ml/g of sucrose solution of packed cells. This suspension was centrifuged at 40,000 g for 1 h. These nuclei were purified by resuspending them in 0.88 M sucrose solution in a ratio of 3 ml/g of solution of packed cells, and centrifuging at 1200 g for 20 min. The nuclei obtained were of low quality because much cytoplasm still adhered to them, but they were satisfactory for obtaining nucleoli. These preparations were resuspended in 2 ml of 0.25 M sucrose solution per I g of packed cells and sonicated with a Branson sonifier. The nucleoli were isolated in the same way as with the liver sonicate. For purification, the nucleoli were suspended in 1 ml of 0.25 M sucrose per 1 g of packed cells, underlayered with 0.88 M sucrose, and the 1 The following abbreviations were used: A, Adenylic acid; C, cytidylic acid; DiVA, deoxyribonucleic acid; G, guanylic acid; i.p., intraperitoneally; i.u., intravenously; 3’-MeDAB, 3’-methyl-4. dimethyl-aminoazobenzene; 4-NQO, 4-nitroquinoline-N-oxide; PCA, perchloric acid; RNA, ribonucleic acid; S, approximate Svedburg coefficient; SDS, sodium dodecyl sulfate; CT,uridylic acid. Experimental

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suspension was centrifuged at 2000 g for 10 min. A satisfactory preparation was usually obtained after this step. Preparation of RNA.-Except for the amount of sodium dodecylsulfate (SDS) solution and phenol used, the procedure for obtaining RNA was the same for all the preparations. For normal liver nucleoli, a ratio of 0.2 : 1, and for regenerating liver nucleoli a ratio of 0.3 : 1 was used of SDS solution and phenol to grams of tissue mass from which nucleoli were derived.’ For Novikoff hepatoma, a ratio of 0.2 ml/g of packed cells was used 1201. The nucleoli were homogenized in a solution containing 0.3 per cent SDS (0.14 M NaCl, and 0.05 M sodium acetate buffer) at pH 5.0; a loose fitting Teflon pestle was used for the homogenization, which was carried out for 1 min (lo-15 strokes). An equal volume of 90 per cent phenol containing 0.1 per cent S-hydroxyquinoline and saturated with the SDS solution was then added and the sample was homogenized again for I min. The suspension was incubated in a water bath at 65°C for 10 min and then was shaken in an Equipoise shaker at room temperature (23%) for 15 min [27]. After the mixture was centrifuged at 17,000 g for 10 min in a Servall centrifuge, the aqueous layer was separated; re-extraction was performed by shaking with an equal volume of the phenol solution for 5 min. After a second centrifugation, the squeous layer was removed and the RNA was precipitated with 3 vol of ethanol containing 2 per cent potassium acetate [27]. Sucrose density gradient centrifugation.-The centrifugation was carried out with a linear gradient of 5-40 per cent sucrose containing 0.1 M NaCl, 1.0 mM EDTA and 0.01 M sodium acetate buffer at pH 5.0-5.1 [22, 281. Approximately 0.5-1.0 ml of RNA solution, containing 0.75-l mg of RNA, was layered on each 26.5 ml gradient and the sample was centrifuged at 15,000 rpm for 15 h in a Spinco SW 25.1 rotor. Fractionation of the gradient was carried out with the aid of an ISCO automatic fractionator (obtained from Instrumentation Specialties, Co., Lincoln, Nebr. [a]. Fractions under each optical density peak were pooled separately and the RNA was precipitated with 3 vol of ethanol containing 2 per cent potassium acetate [27]. In the experiments with nucleolar RNA of regenerating liver, a second purification was made on another gradient. The rapidly sedimenting rapidly-labeled RNA fractions from 3-5 initial gradient runs were pooled and fractionated in lenear gradients of 25-42 per cent sucrose. The additions to the sucrose as well as the force field and duration of the sedimentation were the same as those of the first centrifugation. The purified RNA was precipitated as was the RNA of the first run. Method for quantitation for differences in amount of RNA synthesis.-The areas under the peaks in question were determined with the planimeter (Burrell Corp., Pittsburgh, Pa) an instrument designed for measuring two-dimensional areas. The 6s peak was chosen as a reference peak, since it was the farthest from the peaks in which variations most readily occurred. It gives an indication of the amount of RNA on each gradient. The ratio of the area of the peak to the 6s peak area was d ermined. The same evaluation was made for corresponding peaks in the appropr7 ate control pattern. The differences between these two ratios was determined and then divided by the ratio of the control pattern and multiplied by 100 to determine the 1 Since the RNA content of regenerating liver nucleoli is higher than that of normal nucleoli, a larger volume of SDS solution per gram of tissue mass was used for extracting.

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percent of increase or decrease for a given peak area in question in relation to the corresponding are a of the control. Base analysis.-Hydrolysis of the RNA fractions was carried out with 0.3 N potassium hydroxide at 37°C for 18 h [6]. The hydrolysate was adjusted to pH 6-7 with 0.5 N perchloric acid (PSA) and centrifuged. The supernatant solution was chromatographed on a Dowex-1 formate column for analysis of the nucleotides [12]. After desiccation the nucleotides were dissolved in 0.1 N HCl [17]. An aliquot was taken from each nucleotide fraction for determination of radioactivity and for determination of optical density. Determination of radioactivity.-For the determination of radioactivity distribution in the sucrose density gradients, 0.05 ml of 5.0 N PCA was added to each 1.0 ml fraction of sucrose density gradient, and the samples was hydrolyzed at 70°C for 15 min. To these samples, 8.0 ml of fluor-containing solution [3] were added [27]. For determination of base ratios of RNA by radioactivity, carrier nucleotides were added for chromatography. After chromatography, 10 ml of fluor-containing solution was added to each aliquot from the nucleotide fractions. The radioactivity was determined in an automatic liquid scintillation spectrometer (Packard TriCarb, Series 3000). Electron microscopy.-The tissues were fixed in 2 per cent osmium tetroxide buffered with 0.14 M Verona1 acetate at pH 7.4 for 2 h at 4°C; postfixation was carried out in 10 per cent formalin in 0.1 M phosphate buffer at pH 7.4 for 1 h at 4°C. The fixed tissues were dehydrated with graded concentrations of ethanol containing uranyl acetate. They were embedded in Epon Araldite and stained with 2.5 per cent uranyl acetate in 50 per cent ethanol and 0.2 per cent lead citrate. Thin sections were obtained with an LKB ultrotome and were studied with a Philips EM 200 electron microscope. RESULTS

Effects of aflatoxin

B, on the nucleolar RNA synthesis of normal liver

Fig. 1 presents the sucrose density gradient pattern for nucleolar RNA of normal rat liver, rat liver treated with aflatoxin, B, and rat liver treated with actinomycin D. At 30 min after i.p. injection of 50 pg/kg of aflatoxin B,, the synthesis of 35, 45 and 55s RNA was inhibited. Planimetrically, there was a 45 per cent decrease in relative optical density of the 45s peak. The effect of actinomycin D is shown for comparison. The inhibition is dose dependent as shown in Fig. 2, which shows that the synthesis of 45s RNA is completely inhibited by aflatoxin B, in doses of 200 ,ug/kg or greater. Electron micrographs of the nucleoli of the rat liver treated with 500 ,ug/kg of aflatoxin B, for 1 h showed segregation of the ganular and Iibrillar components (Fig. 3 b). The treated nucleoli were smaller, rounded and more compact than the controls (Fig. 3 a). In the treated nucleoli, areas of densely staining material were observed at the periphery of the nucleoli. The dark material was composed of dense tibrils which were readily distinguishable Experimental Cell Research 51

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from the fibrillar components of the nucleoli. Particles similar in size and density to the granular components were also observed in the dark material. The changes in the nucleoli treated with 50 ,ug/kg of aflatoxin B, were minimal. The nucleoli treated with 150 ,ug/kg of aflatoxin B, showed only segregation of the granular and hbrillar components of the nucleoli.

100 - . . __I50

I 100

0

I I II 200 300 500

Fig. 2.

Fig. 1. Fig. 2.-Effect of dose (log pug/kg), of aflatoxin in nucleoli. Ordinate: Y0 inhibition.

Fig. I.--Sucrose density gradient sedimentation patterns for nucleolar RNA of normal liver. The approximate sedimentation coefficients are shown above the peaks and the shoulders. The distributions of radioactivity following a 10 min pulse of 14C-2-orotic acid are shown as dashed lines. The dose for aflatoxin B, was 50 pg/kg, i.p. while that for actinomycin D was 20 pg/kg, i.v. Abscissa: Fraction number; ordinate: (left) OD at 260 ml”; (right) DPM/fraction. B, (abscissa) on 45s RNA (relative

to 6S RNA)

The 32P base compositions were determined for the 28, 35, 43 and 5% fractions of the nucleolar RNA of normal liver treated with 50 pug/kg of aflatoxin B, for 30 min. The base compositions were not significantly different from those for the corresponding fractions for the control, untreated nucleolar RNA of normal liver (see Table 1). As a drug control, an experiment was done under similar conditions with Fig. 3.-(a) Nucleolus of untreated normal rat liver. The specimen was fixed with osmium tetroxide, followed by 10 % formalin, embedded in Epon Araldite and stained with uranyl acetate and lead citrate. The marker lines in this and the following electron micrographs denote one micron. G, granular components, F, fibrillar components of the nucleolus. x 46,000; (b) Nucleolus of rat liver showing effects of 500 pg/kg of aflatoxin B, at 1 h after administration of the drug. The nucleolus was compact and rounded with segregation of granular (G) from fibrillar (F) components; the presence of dark material (II) is noted. Particles (arrows) were observed inside the dark material. The samples were fixed and stained as indicated for Fig. 3 a. x 45,000. Experimental

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actinomycin D, which produces an increased content of adenylic and uridylic acids in nucleolar RNA [Zl 1. At 30 min after the injection of actinomycin in a dose of 20 ,ug/kg intravenously a 43 per cent decrease occurred in the RNA of the 45 and 55s regions. Table 2 shows that actinomycin D produced a decrease in the guanylic and cytidylic acids of the 55s RNA and a concomittant rise in the adenylic and uridylic acids. TABLE

1. 32P base composition of rapidly sedimenting nucleolar RNA of normal liver, untreated and treated with aflatoxin B, 50 ,ug/kg body weight 28s

35s

45s

55s

19.2 18.5 35.2 27.2

21.2 15.8 38.2 24.8

18.7 16.6 38.1 26.5

20.2 17.3 36.4 26.0

Control Adenine (A) Uracil (U) Guanine (G) Cytosine (C) A+U/GtC

0.61

0.59

0.57

0.60

Treated Adenine (A) Uracil (U) Guanine (G) Cytosine (C) A+U/G+C

TABLE

19.3 18.1 36.6 26.1 0.60

18.3 17.5 37.6 26.6 0.56

18.2 17.3 37.4 27.0

18.4 17.4 37.7 26.5

0.55

0.56

2. 32P base composition of rapidly sedimenting nucleolar RNAof normal liver, untreated and treated with actinomycin D 20 ,ug/kg body weight Treated

Control

A U G c A+U/G+C

Experimental

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45s

55s

45s

55s

18.7 16.6 38.1 26.5

20.2 17.3 36.4 26.0

22.4 21.9 30.0 25.8

22.3 21.8 32.6 23.2

0.57

0.60

0.79

0.79

Effects of aflatoxin

B, and other carcinogens on nucleolar 40-

RLVA

431

CONTROL

3020-

40-

40-

3’- Me DAB

30-

4-N00

302.0 IO-

6

5

Fig. 4.

IO

15

20

25

Fig. G.

I;ig. 4.-Patterns for nucleolar RNA of regenerating liver. Explanation of lettering and symbols is the same as for Fig. 1. The dose of 3’-MeDAB was 100 mg/kg body weight. Abscissa: Fraction number; ordinate: (left) OD at 260 rnp; (right) DPM/fraction. Fig. 6.-Sucrose density gradient sedimentation pattern for RNA of Novikoff hepatoma cells. The explanation of lettering and symbols is the same as for Fig. 1. The dose is 0.5 mg per rat of 4-nitroquionline-N-oxide (4-NQO). Abscissa: Fraction number; ordinate: (left) OD at 250 my; (right) DPM/fraction.

Effects of 3’-methyldimethylaminoazoben.zene RNA synthesis of regenerating liver

(3’-MeDAB)

in the nucleolar

In Fig. 4 (Control) the pattern for nucleolar RNA of untreated 18 h regenerating rat liver is presented. Fig. 4 (3’-MeDAB) shows that at 18 h after injection of 3’-MeDAB in a dose of 100 mg/kg i.p. there was a 64 per cent inhibition of the synthesis of 45 and 55s RNA. There was also a 53 per cent decrease in the labeling of the peak of radioactivity (Fig. 4). Although the TABLE

3. 32P base composition of rapidly sedimenting nucleolar RNA of regenerating liver, untreated and treated with 3-MeDAB 100 mg/kg body weight Control

A u G C A+U/G+C

Treated

45s

55s

45s

55s

19.3 17.9 38.8 24.0

21.3 18.2 36.3 24.1

21.9 16.8 37.8 23.5

21.8 16.9 38.0 23.0

0.59

0.65

0.63

Experimental

0.63

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nucleoli of regenerating rat liver cells were large (Fig. 5 cr), their fine structures were similar to those of normal rat liver nucleoli (Fig. 3 ~1). At 18 h after treatment of the regenerating rat liver x?-ith 3’-MeDAD, the nucleoli were small, rounded and more compact. The fibrillar components of the nucleolus were segregated from the granular components (Fig. 5 b). However, there TABLE 4. 32P base composition of rapidly sedimen tiny nucleolar Xonikoff hepatomn, untreated nnd treated with 4-sQ0

RS.4 of

9.5 mg per rat Control

A U

Treated

45s

55s

11.8 21.7

12.3

12.1

12.4

20.3

20.4

20.7

45s

55s

G

33.7

34.4

34.6

34.1

c

32.8

32.9

32.9

32.8

A:U/G+C

0.50

0.48

0.48

0.49

was no significant difference between the 32P base compositions of the 45 and 55s RNA of the treated and control liver nucleoli of regenerating liver (see Table 3). Effects of 4-nitroquinoline-LV-oxide Xooikoff ascites hepatoma

(4-NQO)

on the nucleolar

RNA synthesis of

In Fig. 6 the sucrose density gradient pattern for nucleolar RNA of untreated Novikoff ascites hepatoma [13] is presented. At 1 h after a dose of 0.5 mg per rat was given i.p., there was a decrease of 32 per cent of the 45 I:ig. 5.-(a) Nucleolus of the regenerating rat liver. Specimens were fixed and stained as for Fig. 3. x 36,000. (h) Nucleolus of the regenerating rat liver treated with 100 mg/kg of 3’.MeDAB for 18 h. The nucleolus was small, rounded and more compact. Fibrillar components (F) were segregated from granular components (G). Specimens were fixed and stained as for Fig. 3. i 33000. Fig. 7.-(a) Electron micrograph of an untreated Novikoff hepatoma cell nucleolus (IVO) fixed and stained as for Fig. 4. x 40,000. (b) Electron micrograph of nucleolus of Novikoff hepatoma cell treated with 5 mg per rat of 4-nitroquinoline-N-oxide for 24 h. The nucleolus was small, rounded and more compact nucleolus with segregation of the fibrillar components (F) from the granular components (G); dark material (S) similar to microspherules [30] was noted. Fixed and stained as indicated for Fig. 3. x 40,000. Fig. 8.-(a) Electron micrograph showing interchromatinic granules (I) in the nucleoplasm of control Novikoff hepatoma cells, fixed and stained as for Fig. 4. x 43,000. (b) Electron micrograph showing focal aggregations of interchromatinic granules in the nucleoplasm of Novikoff hepatoma cell treated as indicated in Fig. 7. Fixed and stained as indicated for Fig. 3. x 43,000. Experimental

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and 55s RNA in the sucrose density gradient patterns. There was also a decrease of 64 per cent in the labeling of the sucrose gradient fraction vvith the maximum radioactivity (Fig. 6, 4-NQO). At 1 h after a dose of 5 mg per rat there was no 33, 45 or 55s RNA in the pattern; also no 3zP vvas incorporated. The 32P base composition of 45 and 55s RNA was not changed from control values by a dose of 0.0 mg of 4-nitroquinoline-N-oxide per rat (see Table 4). Electron micrographs of the treated nucleoli of Novikoff hepatoma cells with 3 mg per rat of 4-NQO for 24 h showed similar changes to those in the nucleoli after treatment with aflatoxin B,. The nucleoli were smaller, rounded and more compact (Fig. 7 b) than those of the control (Fig. 7 a). The granular and fibrillar components of the nucleoli were segregated. The dark material, appearing after 4-NQO treatment, was uniformly small and compact and was similar to the microspherules found in nucleoli of Novikoff hepatoma or Ehrlich ascites cells after treatment with actinomycin D [30]. These spherules in the 4-NQO treated nucleoli seem to be different in its morphology from the dense material found after treatment with aflatoxin B,. In the nucleoplasm, there was aggregation of the interchromatinic granules (Fig. 8 b). After treatment with 0.5 mg of 4-NQO per rat for 1 h there were only minimal alterations in the morphology of nuclei and nucleoli. DISCUSSION

Sporn and Dingman have reported that both aflatoxin B, and 3’-MeDAB bind to DNA and inhibit RNA synthesis [7, 24, 251. 4-NQO has been shown to readily react in vitro with sulfhydryl containing compounds [S, 91 and may thus inhibit RNA synthesis by reacting with enzymes which are involved in RNA synthesis and contain sulfhydryl groups. The present studies show that in the nucleoli of the tissues studied the synthesis of 45s and ~55s RNA is inhibited by these carcinogens. None of these carcinogens caused changes in the 32P base compositions of the rapidly sedimenting RNA. By contrast actinomycin D causes a decrease in the guanylic and cytidylic acids of nucleolar 45 S and 55s RNA’s in normal liver [21]. Actinomycin D is believed to cause this change by specifically binding to deoxyguanosine in DNA and thus interfering with the DNA readout for the synthesis of RNA rich in guanylic and cytidylic acids in particular. Evidently, in the case of aflatoxin B, and 3’-MeDAB, the effect on DNA differs in as much as there is no specific change in nucleotide composition of the newly synthesized RNA. There are no reports yet that 4-nitroquinoline-N-oxide binds to DNA. Electron microscopic studies showed that there were morphological changes Experimental

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in nucleoli treated with aflatoxin B,, 3’-MeDAB and 4-NQO. These changes (segregation and decrease in nucleolar size) are quite similar to those seen with actinomycin D [21, 291 and have been reported previously by Bernhard et aZ. [l] and Lazarus [16]. In the case of 4-NQO, focal aggregations of interchromatinic granules vvere seen in the extranucleolar regions. These may be the nuclear inclusion bodies seen by light microscopy by Endo et al. [S, 91. Actinomycin D, aflatoxin B,, 4-NQO and 3’-MeD,4B cause decreases in nucleolar size, but the mechanism is not known. Electron micrographs show that granular plaques appear to be migrating away from nucleoli treated with 4-NQO. Svoboda et at. [29] published a similar picture, showing dark granular material apparently moving away from a compact nucleolus of rat liver that was treated with aflatoxin B,. The nucleoli may decrease in size due to loss of material into the nucleoplasm. However, since these compounds have been shown to be potent inhibitors of RNA synthesis in nucleoli, the depletion of nucleolar RNA may result from normal loss of nucleolar products while there is no replacement. Preliminary studies in this laboratory showed that, in the nucleoli of Novikoff hepatoma, actinomycin D caused an inhibition of RNA synthesis and a biosynthesis of RNA that is less rich in guanylic and cytidylic acids and more abundant in adenylic and uirdylic acids. This effect is the same as that noted with normal liver treated with actinomycin D. Actinomycin D also causes similar nucleolar changes in morphology [al] as seen with aflatoxin B,, 3-MeDAB and 4-NQO, but there have not been reports of carcinogenicity with this compound. It is interesting to speculate that the carcinogenic activity of aflatoxin B, 3’-MeDAB and 4-NQO may be related to their nonspecific effects on nucleolar RNA synthesis. The changes produced by actinomycin D (decrease in guanylic and cytidylic acids and increase in adenylic and uridylic acids), are the opposite changes expected for carcinogenic effects because studies in this laboratory have shown that the base compositions of rapidly sedimenting nucleolar RNA of several different cancer tissues are richer in guanylic and cytidylic acids and lower in adenylic acid [26] in comparison with the corresponding RNA of normal liver [IT]. SUMMARY

Studies were made on the acute effects of aflatoxin B,, 3’-methyl-4-dimethylaminoazobenzene, and 4-nitroquinoline-N-oxide on nucleolar RNA synthesis of normal liver, regenerating liver, and Novikoff hepatoma. Sedimentation profiles obtained from density gradient centrifugation of the nucleolar RNA of these tissues showed that these carcinogens inhibited the Experimental

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L. R. Floyd,

T. Unumcr und H. Busch

synthesis of rapidly sedimenting nucleolar RNA and electron microscopic studies showed that each produced segregation of nucleolar granular and hbrillar elements. Unlike results with actinomycin D, which produces similar morphological effects, the 32P base compositions of the rapidly sedimenting nucleolar RNA showed no changes in comparison with base compositions of corresponding fractions of untreated tissues. Electron microscopic studies showed that aflatoxin B,, 3’-MeDAB and 4-nitroquinoline-N-oxide had similar compact nucleoli, a decrease in nucleolar size, segregation of the fibrillar from granular components. Dense material in the nucleoli \\-as produced by aflatoxin B, and by +nitroquinoline-N-oxide. These results show that any substance that inhibits nucleolar biosynthesis also produces segregation of nucleolar granular and hbrillar elements. We would like to express our appreciation to Helen Adams and Dr Ken Higashi for their valuable technical advice. We also thank Dr Charles M. Mauritzen for his helpful instruction on the synthesis and administration of 3’-MeDAB. REFERENCES 1. BERNHARD, W., FRAYSSINET, C., LAFARGE, C. and LE BRETON, E., Compf. Rend. Acad. Sci. 261, 1785 (1965). 2. BRAKKE, M. K., Anal. Biochem. 5, 271 (1963). 3. BRUNO, G. A. and CHRISTIAN, J. E., Anal. Chem. 33, 1216 (1961). 4. BRYAN, R. W., J. Natl. Cancer Inst. 24, 221 (1960). 5. ~ Texas Rept Biol Med. 15, 674 (1957). 6. DAVIDSON, J. N. and SMELLIE, R. M. S., Biochem. J. 52, 594 (1952). 7. DINGMAN, C. W. and SPORN, M. B., Cancer Res. 27, 938 (1967). 8. ENDO, H., Gann 49, 151 (1958). 9. ENDO, H., AOKI, M. and AOYAMA, Y., Cann 50, 209 (1959). 10. FUKUOKA, F. and NAORA, H., Gann 48, 271 (1957). 11. GIESE, J. E., MILLER, J. A. and BAUYANS, C. A., Cancer Res. 5, 337 (1945). 12. HURLBERT, R. N., SCHMITZ, H., BRUMM, A. F., and POTTER, V. R., J. Biot. Chem. 209, 23 (1954). 13. KAMAHORA, J. and KAHUKAGA, T., Proc. Japan Acad. 42, 1079 (1966). 14. KIZER, D. E., HOWELL, B. A., SHIRLEY, B. C., CLOUSE, .J. E. and Cox, B., Cancer Res. 26, 822 (1966). 15. LANCASTER, M. C., JENKINS, F. P., and PHILIP, J. McL., Nature 192, 1095 (1961). 16. LAZARUS, S. S., VETHAMANY, V. G., SHAPIRO, S. H. and AMSTERDAM, D., Cancer Res. 26, 2229 (1966). 17. MURAMATSU, M. and BUSCH, H., Cancer Res. 24, 1028 (1964). 18. NAKAHARA, W. and FUKUOKA, F., Gann 50, 1 (1959). 19. NAKAHARA, W., FUKUOKA, F. and SUGI~IURA, T., Gann 48, 129 (1957). 20. NAKAMURA, T., RAPP, F. and BUSCH, H., Cancer Res. 27, 1084 (1967). 21. Ro, T. S., SHANKAR NARAYAN, K. and BUSCH, H., Cancer Res. 26, 780 (1966). 22. SCHERRER, K. and DARNELL, J. E., Biochem. Biophys. Res. Commun. 1, 486 (1962). 23. SIMARD, R. and BERNHARD, W., Intern. J. Cancer 1, 463 (1966). 24. SPORN, M. B. and DINGMAN, C. W., Cancer Res. 26, 2488 (1966). 25. SPORN, M. B., DINGMAN, C. W., PHELPS, H. L. and WOGAN, G. N., Science 151, 1539 (1966). 26. STEELE, W. J. and BUSCH, H., Biochim. Biophys. Acta 119, 501 (1966). 27. STEELE, W. J., OKAMURA, N. and BUSCH, H., Biochim. Biophys. Acta 87, 490 (1964). 28. ~ J. Btot. Chem. 240, 1742 (1965). 29. SVOBODA, D., GRADY, H. and HIGGIXSOS, J., Am. J. Path. 49, 1023 (1966). 30. UNUMA, T. and BUSCH, H., Cancer Res. 27, 1232 (1967).

Experimental

Cell Research 51