effect of nitrofurans antagonistic to 3'-methyl-4 ......polymerase of liver nuclei (chart 1)....

[CANCER RESEARCH 34, 1843-1850, August 1974]

Effect of Nitrofurans Antagonistic to3'-Methyl-4-dimethylaminoazobenzene in Hepatocarcinogenesis andRNA Polymerase Activity of Liver Cell Nuclei in Rats1

Mitsutaro Akao, Keiko Kuroda, Yoshihiro Tsutsui.2 Masayoshi Kanisawa, and Komei Miyaki

Research Institute for Chemobidynamics, Chiba University, Izumi-cho, Narashino, Chiba 275 [M. A., K. K., M. K., K. M.], ana Departmentof Pathology, Faculty of Medicine, Nagoya University, Showa-ku, Nagoya, A Â¡chi466 [Y. 7".]2,Japan

SUMMARY

The purpose of this study was to determine (a) whetherthe hepatocarcinogenesis in rats fed 3'-methyl-4-dimeth-ylaminoazobenzene (3'-Me-DAB) could be inhibited by

the simultaneous and followed feeding of two nitrofurans;(b) whether 3'-Me-DAB feeding inhibited DNA-dependent

RNA polymerase activity of liver nuclei; and (c) whether thenitrofurans ameliorated 3'-Me-DAB-induced changes inRNA polymerase activity. The nitrofurans were 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide and 2-amino-5-[2-(5-nitro-2-furyl)-l-(2-furyl)vinyl-l-]-l ,3,4-oxadiazole. MaleDonryu rats were fed 1 or 0.5 g of 3'-Me-DAB by beingmaintained on a diet containing 0.06% 3'-Me-DAB or3'-Me-DAB plus 0.2% of a nitrofuran, and then they were

maintained on basal diet for 60 or 300 days, respectively.The incidence of hepatomas induced by 3'-Me-DAB wasreduced to about one-fifth by the simultaneous feeding of anitrofuran. Feeding of 3'-Me-DAB brought gradual reduc

tions of RNA polymerase activities of liver nuclei; 65 to80% reduction of Mn' --(NH4)2SO4-activated RNA polymerase activity and 50% reduction of the Mg+*-activated onewere observed in the rats fed 3'-Me-DAB diet for more than

27 days. The reduction of RNA polymerase activity wasdelayed or prevented by the simultaneous feeding of anitrofuran. The induction of hepatomas in rats fed 0.5 g3'-Me-DAB was also retarded by the followed feeding of a

nitrofuran. The followed feeding of a nitrofuran promotedthe recovery of 3'-Me-DAB-induced deleterious changes in

RNA polymerase activity and liver nucleic acid contents.These and associated findings indicated that the nitrofuransretarded 3'-Me-DAB carcinogenesis by exhibiting their owneffect, antagonistic to 3'-Me-DAB, on rat liver.

INTRODUCTION

rats for 10 days at a level of 0.06% in the diet, causedinhibition of DNA-dependent RNA polymerase (EC2.7.7.6) activity of liver nuclei. In order to provide furtherevidence for the assumption that carcinogenic azo dyescause inhibition of RNA polymerase activity of liver nuclei,the present study was made on 3'-Me-DAB, because3'-Me-DAB is more potent in the carcinogenic activity than

DAB and causes nucleolar segregation of liver nuclei (12,18, 19), which is well associated with inhibition of RNApolymerase activity of liver nuclei specifically with regard toaflatoxin B!, one of the most potent hepatocarcinogens (13,19, 20, 23, 24).

Another purpose of this study is to provide someinformation that might be useful for assessing whetherinhibition of RNA polymerase activity is an importantevent in azo dye hepatocarcinogenesis. In this respect, it isworthwhile to follow RNA polymerase activity throughoutthe carcinogenic process and also to examine whether RNApolymerase activity would be influenced by the substancesthat inhibit azo dye carcinogenesis. As such inhibitors, wechose Nl and N2 for this study, because either nitrofuranexhibits a strong inhibitory effect on DAB carcinogenesiswhen it was fed to rats simultaneously with DAB (1, 10, 11).The 3rd purpose is to demonstrate the possibility that thesenitrofurans would inhibit azo dye carcinogenesis by exhibiting their own effect on rat liver, because the nitrofurans, incontrast to DAB, stimulate RNA polymerase activity ofliver nuclei. As an approach to demonstrate this possibility,studies were also made to determine whether the hepatocarcinogenesis induced by 3'-Me-DAB could be inhibited not

only by the simultaneous feeding of the nitrofurans but alsoby the followed feeding of them and then to inquire whethersuch aspects in the inhibition of hepatocarcinogenesis wouldbe reflected on RNA polymerase activity of liver nuclei.

In the previous study (2), we found that DAB,3 fed to the MATERIALS AND METHODS

1Supported by a Grant-in-Aid for Scientific Research from theJapanese Ministry of Education.

â€¢¿�'Present address: Aichi Cancer Research Institute, Chigusa-ku, Na

goya, Aichi 464, Japan.3The abbreviations used are: DAB, 4-dimethylaminoazobenzene;

3'-Me-DAB, 3'-methyl-4-dimethylaminoazobenzene; Nl, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; N2, 2-amino-5-[2-(5-nitro-2-furyl)-1-(2-furyl(vinyl-1-1-1,3,4-oxadiazole.

Received February 9, 1974; accepted April 10, 1974.

Animals used were male and female Donryu rats, obtained from Nippon Rat Co., Saitama, Japan. They weremaintained on a semisynthetic diet, CE-2 (Ref. 11; consisting of protein, 24%; fat, 3.5%; carbohydrate, 56%;minerals, 6%), obtained from CLEA Japan Inc., Tokyo,Japan. At 8 to 9 weeks of age, they were divided into groupsand fed one of the experimental diets, namely 3'-Me-DAB

AUGUST 1974 1843

Research. on October 23, 2017. © 1974 American Association for Cancercancerres.aacrjournals.org Downloaded from

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M. Akao et al.

diet (CE-2 diet containing 0.06% 3'-Me-DAB), 3'-Me-DABplus Nl diet (3'-Me-DAB diet containing 0.2% N1), 3'-Me-DAB plus N2 diet (3'-Me-DAB diet containing 0.2% N2).

Autopsies were performed on both the animals dead inthe course of feeding experiments and those sacrificed at theend of the feeding schedules. Tissue samples for microscopywere fixed in 10% formaldehyde solution, and paraffinsections were stained with hematoxylin and eosin. Forelectron microscopy, tissue samples were minced into piecesof about 1 cu mm, fixed in 2% glutaraldehyde for 2 hr, andpostfixed with 1%OsO4 for 3 hr. They were then dehydratedin a graded series of alcohol and propylene oxide andembedded in Epon 812. Sections were double stained withuranyl acetate and lead citrate and examined with a HitachiHU-11DS electron microscope.

Isolation of liver nuclei was made by a modification of themethod of Dingman and Sporn (3); details of the procedureare described in the previous report (2). The mean RNA/DNA ratio of the isolated liver nuclei from the animals fedbasal diet was 0.17, and the recovery of the nuclei in thenuclear fraction from the liver homogenate was more than50%, based on DNA analysis. RNA polymerase activity ofthe isolated liver nuclei was assayed in the systems underactivation by Mg+ + at low ionic strength and by Mn++ at

high ionic strength with ammonium sulfate, according to theprocedure of Widnell and Tata (21, 22). Radioactivity wasmeasured in Beckman LS-150 liquid scintillation systemand the extent of quenching was estimated by the internalstandardization technique using a toluene-14C standard. The

mean efficiency of counting was 52%.The quantity of protein bound dye was determined

according to the method of Miller and Miller (8). The

quantitative analyses of RNA and DNA were made by themethods of Fleck and Begg (4) and Giles and Myers (5),respectively. Protein was analyzed according to the methodof Lowry et al. (7), with crystalline bovine albumin asstandard. UTP-4-'*C was obtained from the Radiochemical

Centre, Amersham, England; and GTP, CTP, ATP, andUTP were obtained from BÃ¶ehringerund SÃ¶hneGmbH,Mannheim, Germany. Nitrofurans were donations from theUeno Pharmaceutical Co., Ltd., Osaka, Japan.

RESULTS

The effect of the simultaneous and followed feeding on3'-Me-DAB hepatocarcinogenesis was examined in rats fed1 or 0.5 g of 3'-Me-DAB and is summarized in Table 1. Ratsof Groups 1A to 1C' were fed an experimental diet,described in "Materials and Methods," until they consumed1 g of 3'-Me-DAB or l g of 3'-Me-DAB and 3.34 g of a

nitrofuran. This required about 100 days; thereafter, eachgroup was fed basal diet for 60 days (male) or 90 days(female). Ten of 11 male rats fed 3'-Me-DAB alone (Group

1A) developed hepatomas. The simultaneous feeding of anitrofuran with 3'-Me-DAB reduced the incidence of hepa

tomas to 2 among 11 animals (Groups IB and 1C). Femalerats were somewhat resistant to the carcinogenic activity of3'-Me-DAB. Hepatomas were found in 5 of 12 femaleanimals fed 3'-Me-DAB alone (Group 1A') and noneamong 10 animals fed 3'-Me-DAB plus N2 diet (Group1C'). Rats of Groups 2A, 2B, and 2C were fed 0.5 g3'-Me-DAB or 0.5 g 3'-Me-DAB and 1.67 g of a nitrofuran

by being maintained on an experimental diet. Rats of

Table 1Effect of simultaneous and followed feeding of a nitrofuran on induction of hepatoma and associated liver

lesions in the rats fed I or 0.5 g of Ã¬'-Me-DABMale and female rats of Groups IA to 1C' were fed l g of 3'-Me-DAB by being maintained on diet

containing 0.06% 3'-Me-DAB or 3'-Me-DAB plus 0.2% of a nilrofuran. Thereafter, each group was fed basaldiet for 60 days (male) or 90 days (female). Male rats of Groups 2A to 2E were fed 0.5 g of 3'-Me-DAB by

being maintained on an experimental diet described above. The rats of Groups 2D and 2E were first fed 0.5 g of3'-Me-DAB, and then they were fed 1.67 g of a nitrofuran by being maintained on diet containing 0.2% of a

nitrofuran. Thereafter, each of Groups 2A to 2E was maintained on basal diet and sacrificed at 1year alter thestart of feeding with experimental diets.

Intake(g/rat) of Associated liver lesions

GroupIAIB1C1A11C2A2B2C2D2ENo.andsex

ofrats11

MIIM11M12FIO

FII

M4M8M5MII

M3'-Me-DAB0.50.50.50.50.5NitrofuranNone3.34

(NI)3.34(N2)None3.34

(N2)None1.67

(Nl)1.67(N2)I.67(NI")1.67(N2Â°)Incidence

ofhepatomaIO2250IO1112AdenomaandidenomatousgrowthII653434424Cholangio-fibrosis10403050034

' Followed feeding of a nitrofuran.

1844 CANCER RESEARCH VOL. 34



Groups 2D and 2E were first fed 0.5 g 3'-Me-DAB, and then

they were fed 1.67 g of a nitrofuran by being maintained onbasal diet containing 0.2% nitrofuran. Thereafter, all groupswere maintained on basal diet and sacrificed 1year after thestart of feeding with experimental diets. Feeding of 0.5 g3'-Me-DAB was enough for the induction of hepatomas inmale rats, and 10 of 11 animals fed 3'-Me-DAB alone

(Group 2A) developed hepatomas. The incidence of hepatomas was reduced not only by the simultaneous feeding of anitrofuran (Groups 2B and 2C) but also by the followedfeeding of a nitrofuran (Groups 2D and 2E).

Feeding 3'-Me-DAB to male rats brought about reduction of 2 kinds of activities of DNA-dependent RNApolymerase of liver nuclei (Chart 1). Mgv*-activated RNA

polymerase activity was inferred to be associated with thesynthesis of rRNA, and the Mn+"-(NH4)2SO4-activated

one was inferred to be associated with the synthesis ofDNA-like RNA (22). The reduction of Mn+ +-(NH4)2SO4-

activated RNA polymerase activity was more remarkable;70 to 80% reduction was seen in the rats fed 3'-Me-DAB dietfor 27 and 50 days, while the reduction of the Mg++-

Chart I. Effect of the feeding of 3'-Me-DAB and a nitrofuran on RNA

polymerase activities of liver nuclei. Each point represents the mean of theenzyme activities of 4 rats. The value of the rats fed basal diet is madecontrol (100%) and the value of the rats fed 3'-Me-DAB or 3'-Me-DAB anda nitrofuran is expressed in as percentage Â±S.E. â€¢¿�,fed 3'-Me-DAB: A.fed 3'-Me-DAB plus Nl diet: A, fed 3'-Me-DAB plus N2 diet. In nucleifrom the control rats fed basal diet, the en/yme activity for Mg+^-activated

system was 600 to 700 pmoles UTP incorporated into RNA per mg DNA;for Mn* -(NH,)2SO,-activated system it was 1600 to 1800 pmoles UTP

per mg DNA.

Nitrofurans against Azo Dye Carcinogenesis

activated RNA polymerase activity was about 50%. Thesimultaneous feeding of a nitrofuran brought an elevation ofMg* '-activated RNA polymerase activity, which was seenat 7 and 15 days in the animals fed 3'-Me-DAB plus N l (or

N2) diet, and prevented this RNA polymerase activity frombeing reduced by 3'-Me-DAB at 27 and 50 days. On theother hand, the effect of 3'-Me-DAB on Mn+ MNH4)2SO4-

activated RNA polymerase activity was so strong that thesimultaneous feeding of a nitrofuran prevented the activityreduction only during the early days (7 and 15 days) offeeding. In Table 2 are summarized the repeated experiments on RNA polymerase activities and liver nucleic acidcontents with 10 animals fed one of the experimental dietsfor 50 days. In agreement with the findings in RNApolymerase activities, 3'-Me-DAB feeding reduced RNA/

DNA ratios of liver and liver nuclei. The simultaneous

50,'0 7 15 Â«)DAYS PP FOLLOWED FEEDING

0 7 15DAY? OF FOLLOWFD FEEDING

Chart 2. Effect of the followed feeding of a nitrofuran (N2) on therecovery of RNA polymerase activities of liver nuclei. Male rats were fed0.06% 3'-Me-DAB diet for 50 days, and then they were followed to be fed

basal diet or diet containing 0.2% N2 for 7, 15,and 40 days. Each pomi onthe graph represents the mean of the enzyme activities of 4 rats (7 and 15days) or 10 rats (0 and 40 days) and is expressed as percentage Â±S.E. ofthe control value from the rats fed basal diet throughout the feedingschedule. The values of Day 0, namely, the enzyme activities in the ratsfed 3'-Me-DAB diet for 50 days, are obtained from Table 2. â€¢¿�,followed

feeding with basal diet: A, followed feeding with diet containing 0.2% of anitrofuran (N2).

Table 2RNA polymerase activities of isolated liver nuclei and contents of liver nucleic acids in male rats fed one of the experimental

diets for 50 days

RNA polymerase activity(pmoles UTP/mg DNA) RNA/DNA ratio

Diet"Basal

3'-Me-DAB3'-Me-DAB + Nl3'-Me-DAB + N2Mg"-

activated666

Â±102"r337Â± 77

596 Â±18K722 Â±205CMn++-(NH4),SO4-activated1760

Â±376'637 Â±301603 Â±378850 Â±568Liver

nuclei0.171

Â±0.009a0.154 Â±0.0210.204 Â±0.022f0.204 Â±0.021'Liver4.08

Â±0.41'1.97 Â±0.952.88 Â±0.51"3.09Â±0.71CLiver

DNA(mg/gliver)l.85Â±0.13r

3.12 Â±0.41l.87Â±0.33f2.01 Â±0.40e

a3'-Me-DAB diet, diet containing 0.06% 3'-Me-DAB; 3'-Me-DAB + Nl diet, 3'-Me-DAB diet containing 0.2% Nl; 3'-Me-DAB + N2 diet, 3'-Me-DAB diet containing N2.

" Mean of the values for 10 animals Â±S.D.' p < 0.01, relative to the rats fed 3'-Me-DAB diet.dp < 0.05, relative to the rats fed 3'-Me-DAB diet.

AUGUST 1974 1845



M. Akao et al.

Table 3Effect of followed feeding of a nilrofuran on recoveries ofRNA polymerase activities of isolated liver nuclei and contents of liver

nucleic acids in male rats fed i'-Me-DABMale rats were fed diet containing 0.06% 3'-Me-DAB for 50 days, and then they were divided into 2 groups. One group was

further maintained on basal diet and the other group was on the diet containing 0.2% N2 for 40 days.

RNA polymerase activity(pmolesUTP/mg DNA) RNA/DNA ratio

Group"Basal

3'-Me-DAB -basal3'-Me-DAB -N2Mg*'-activated706

Â±102"

644 Â±105959 Â± 95rMn"-(NH4)2SO4-

activated1822

Â±28Â»1480 Â±2331784 Â±322"Liver

nucleiO.I

70 Â±0.0210.157 Â±0.0320.223 Â±0.030eLiver4.02

Â±0.28'

3.39 Â±0.293.93 Â±0.24'Liver

DNA(mg/gliver)1.93

Â±0.16'2.40 Â±0.062.03 Â±0.13'

" Basal, fed basal diet alone; 3'-Me-DAB â€¢¿�basal, fed basal diet following ingestion of 3'-Me-DAB; 3'-Me-DAB â€¢¿�N2, fed dietcontaining 0.2% of a nitrofuran (N2) following ingestion of 3'-Me-DAB.

" Mean of the values for 10 animals Â±S.D.' p < 0.01, relative to 3'-Me-DAB â€¢¿�basal group."p < 0.05, relative to 3'-Me-DAB â€¢¿�basal group.

feeding of a nitrofuran prevented the reduction of liverRNA/DNA ratio and elevated the RNA/DNA ratio ofliver nuclei. The reductions of RNA polymerase activitiesand liver RNA/DNA ratio associated with the ingestion of3'-Me-DAB were recovered by the followed maintenance of

the rats on basal diet (Chart 2). The followed feeding of anitrofuran promoted the recovery of RNA polymeraseactivities (Chart 2) and the levels of liver nucleic acidcontents (Table 3).

The effect of nitrofurans against 3'-Me-DAB was also

noted in the histological examinations of liver. The markedproliferation of ductal cells caused by 3'-Me-DAB was

effectively reduced by the simultaneous feeding of a nitrofuran (Figs. 1 to 3). The condensation and local segregation orlight capping of the nucleolus were often observed in theliver cells of the rats fed 3'-Me-DAB diet (Fig. 4). Either

nitrofuran caused a hypertrophy of the nucleolus (Fig. 6)and preserved the loose structure of the nucleolus againstthe attack of 3'-Me-DAB (Fig. 5). It is conceivable that

nitrofurans would exhibit influences on the metabolic fate of3'-Me-DAB and prevent the carcinogen from interacting

with the constituents of liver. Therefore, the level of theprotein-bound dye in the liver was estimated as a measure ofthe interaction of 3'-Me-DAB with liver constituents (Chart

3). However, the simultaneous feeding of a nitrofuran with3'-Me-DAB brought no significant effect on the level of theprotein-bound dye.

DISCUSSION

Feeding 3'-Me-DAB to rats brought reductions of RNA

polymerase activities of liver nuclei, and the reduction ofMn ' ^-(NH4)2SO4-activated one was more remarkable thanthat of Mg+ +-activated one. Such selectivity in the reduction of RNA polymerase activity was more clearly indicatedin our previous observation on DAB (2). Kizer and Clouse(6) presented a similar finding that the rats fed 3'-Me-DABshowed decreased incorporation of orotic acid-6-14C into

nonnucleolar nRNA, but incorporation into nucleolar RNAwas decreased to a lesser extent. The reduction of RNA

1.25

1.00

0.75

0.50

0.25

0.007 15 27 50

DAYS OF FEEDING WITH EXPERIMENTAL DIET

Chart 3. Effect of the simultaneous feeding of a nitrofuran on theprotein-bound dye level in the liver of male rats fed 3'-Me-DAB. Each

point represents the mean of the protein-bound dye levels of 4 rats,estimated by the absorbance at 520 nm of the acid solution of polar dyefrom 50 mg liver protein. The mean values of the control rats fed3'-Me-DAB diet for 7, I5, 27, and 50 days were 0.254, 0.261, 0.197, and0.184, respectively. The values of the rats fed 3'-Me-DAB plus a nitrofuran

diet are expressed in fractions of the control values Â±S.D. A, ted3'-Me-DAB plus NI diet; A, fed 3'-Me-DAB plus N2 diet.

polymerase activities was accompanied by the reduction ofRNA/DNA ratios of liver and liver nuclei (17) and alsowith nucleolar segregation of liver cell nuclei (12, 18, 19).These effects of 3'-Me-DAB were stronger than those ofDAB (2) in agreement with the fact that 3'-Me-DAB is

stronger in carcinogenic activity than is DAB (9).The simultaneous feeding of the present nitrofurans

delayed or retarded the induction of hepatomas in rats fed





3'-Me-DAB.4 These nitrofurans, in contrast to the azo dye

carcinogens, can enlarge rat liver and stimulate RNApolymerase activities of liver nuclei. These effects andactivities of the nitrofurans were also noted in the rats fed3'-Me-DAB and a nitrofuran simultaneously, and they

reflected on RNA polymerase activities, fine structures ofliver nuclei, and the contents of liver nucleic acids. On theother hand, the level of the protein-bound dye, which wasestimated as a measure of the reaction of 3'-Me-DAB with

liver constituents, were not significantly influenced by thesimultaneous feeding of a nitrofuran. Therefore, it isassumed that the nitrofurans retarded the induction ofhepatomas by exhibiting their own effects, antagonistic to3'-Me-DAB, on rat liver. This assumption was further

confirmed by the finding that the followed feeding of anitrofuran could also retard the induction of hepatomas inthe rats fed 3'-Me-DAB, promoting the recovery of RNA

polymerase activities and the levels of liver nucleic acids.The inhibition of RNA polymerase activity of liver nuclei

has been well associated with nucleolar segregation in somehepatotoxins such as aflatoxin B, and actinomycin D (13,15, 23). Therefore, the reduction of RNA polymeraseactivities associated with the ingestion of 3'-Me-DAB canbe attributed to the toxic effect of 3'-Me-DAB on liver cells.

However, there remains the possibility that the reduction ofRNA polymerase activities was also due to the changes inliver cell population, especially in the late stage of azo dyefeeding (14, 16). Detailed studies are necessary to follow thefate of the cells that were attacked by 3'-Me-DAB and todetermine the meanings of the effect of 3'-Me-DAB and the

present nitrofurans on RNA polymerase activities of livernuclei in the processes of hepatocarcinogenesis.

REFERENCES1. Akao, M., Kuroda. K., Kanisawa, M., and Miyaki, K. Influence of

Some Nitrofurans on Carcinogenesis in Rats Fed 4-(Dime-thylamino)azobenzene. Gann, 62: 479-484, 1971.

2. Akao, M., Kuroda, K., and Miyaki, K. Selective Inhibition of RNAPolymerase Activity in Rat Liver Nuclei by 4-(Dime-thylamino)azobenzene, and Effect of Nitrofurans on Liver RNAMetabolism Associated with Prevention of Carcinogenesis. Gann, 63:1 10, 1972.

3. Dingman, C. W., and Sporn. M. B. Studies on Chromatin I. Isolationand Characterization of Nuclear Complexes of DeoxyribonucleicAcid, Ribonucleic Acid, and Protein in Embryonic and Adult Tissuesof the Chicken. J. Biol. Chem., 239: 3483 3492, 1964.

4. Fleck, A., and Begg. D. The Estimation of Ribonucleic Acid UsingUltraviolet Absorption Measurement. Biochim. Biophys. Acta, 108:333 339, 1965.

5. Giles, K. W., and Myers, A. An Improved Diphenylamine Method forthe Estimation of Dexoyribonucleic Acid. Nature, 206: 93, 1965.

6. Kizer, D. E., and Clouse, J. A. Effect of Hepatocarcinogens on the

4The histological examination of rat livers indicated that adenoma or

adenomatous growth of hepatocytes was frequently found in the livers ofthe rats fed both 3'-Me-DAB and a nitrofuran (Table I). Thus, theprotection of a nitrofuran from 3'-Me-DAB-induced neoplastic transfor

mation was consistently exerted only against hepatoma induction.

Synthesis and Content of Rat Liver Nuclear RNA. Cancer Res., 28:502-509, 1968.

7. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall. R. J.Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem.,193: 265-275, 1951.

8. Miller, E. C., and Miller, J. A. The Presence and Significance ofBound Aminoazo Dyes in the Liver of Rats Fed p-Dime-thylaminoazobenzene. Cancer Res., 7: 468 480, 1947.

9. Miller, J. A., and Miller, E. C. The Carcinogenic Aminoazo Dyes.Advan. Cancer Res., /: 339-396, 1953.

10. Miyaji, T. The Effect of Simultaneous and Alternate Feeding ofFurylfuramide and 4-Dimethylaminoazobenzene on Rats. Tohoku J.Exptl. Med.. W3: 371 379, 1971.

11. Miyaki, K., Akao, M., Terao, K., and Kuroda, K. Modes of Action ofThree Bacteriostatic Agents in Their Inhibitory Effect on Induction ofHepatoma in Rats Fed 4-(Dimethylamino)-azobenzene. I. Effect onMetabolic Fate of 4-(Dimethylamino)-azobenzene. Gann, 60:167-179, 1969.

12. O'Hegarty, M. T., and Harman, J. W. The Inhibitory Effect of

3-Methylcholanthrene on Nucleolar Alterations Induced in Rat LiverCells by 3'-Methyl-4-dimethylaminoazobenzene. Cancer Res., 29:

521 528, 1969.13. Pong, R. S., and Wogan, G. N. Time Course and Dose-Response

Characteristics of Aflatoxin B, Effects on Rat Liver RNA Polymeraseand Ultrastructure. Cancer Res., 30: 294 304, 1970.

14. Rabes, H. M., Hartenstein, R., and Ringelmann, W. DNA Synthesisin Rat Liver during Early Stages of Azo Dye-induced Hepatocarcinogenesis. Cancer Res., 32: 83 89, 1972.

15. Schwartz, H. S., Sternberg, S. Sâ€žand Philips, F. S. SelectiveCytotoxicity of Actinomycin in Mammals. In: S. A. Waksman (ed.),Actinomycin, pp. 101-121. New York: John Wiley and Sons, Inc.,1968.

16. Sneider, T. W., Bushnell, D. E., and Potter, V. R. The Distributionand Synthesis of DNA in Two Classes of Ral Liver Nuclei during AzoDye-induced Hepatocarcinogenesis. Cancer Res.. 30: 1867-1873,1970.

17. Sporn, M. B., and Dingman, C. W. Studies on Chromatin II. Effect ofCarcinogens and Hormones on Rat Liver Chromatin. Cancer Res., 26:2488 2495, 1966.

18. Svoboda, D., and Higginson, J. A Comparison of UltrastructuralChanges in Rat Liver due to Chemical Carcinogens. Cancer Res., 28:1703-1733, 1968.

19. Svoboda, D., RÃ¡cela,A., and Higginson, J. Variations in Ultrastructural Nuclear Changes in Hepatocarcinogenesis. Biochem. Phar-macol., 16: 651 657, 1967.

20. Troll, W., Belman, S., Berkowitz, E., Chmielewicz, Z. F., Ambrus, J.L., and Bardos, T. J. Differential Responses of DNA and RNAPolymerase to Modification of the Template Rat Liver DNA Causedby Action of the Carcinogen Acetylaminofluorene In Vivo and InVitro. Biochim. Biophys. Acta, 157: 16 24, 1968.

21. Widnell, C. C., and Tata, J. R. A Procedure for the Isolation ofEnzymatically Active Rat-Liver Nuclei. Biochem. J., 92: 313-317,1964.

22. Widnell, C. C., and Tata, J. R. Studies on the Stimulation byAmmonium Sulfate of the DNA-Dependent RNA Polymerase ofIsolated Rat-Liver Nuclei. Biochim. Biophys. Acta, 123: 478 492,1966.

23. Wogan, G. N. Biochemical Responses to Aflatoxins. Cancer Res., 28:2282-2287, 1968.

24. Wogan, G. N., and Newberne, P. M. Dose-Response Characteristicsof Aflatoxin B, Carcinogenesis in the Rat. Cancer Res., 27:2370-2376, 1967.

AUGUST 1974 1847



M. Akao et al.

Figs. I to 3. Micrographs of livers of male rats fed an experimental diet for 24 days. P. peripheral parts: C, central veins. H & E, x 80.Fig. I. Liver of a rat fed _V-Me-DABdiet. The extensive proliferation of ductal cells is seen in the peripheral parts of liver lobule. The hepatocytes

around the central veins are hypertrophie and degenerative.Fig.2. Liver of a rat fed 3'-Me-DAB plus Nl diet. The anisocytosis and atrophy of hepatocytes are seen. However, the proliferation of ductal cells is

not evident.Fig. 3. Liver of a rat fed 3'-Me-DAB plus N2 diet. A finding similar to that of Fig. 2.

Figs. 4 to 6. Electron micrographs of liver cell nuclei of male rats fed an experimental diet for 24 days. Bar. I ^m. Uranyl acetate and lead citrate, x13,000.

Fig. 4. Liver nucleus of a rat fed 3'-Me-DAB diet. The nucleoli are small. An assembly of granular components (G) is seen at the center of the

nucleolus at the righi side. The nucleolus at the left side exhibits a light nucleolar cap (Â¿C).The chromatin around the nucleolus is diffuse and thin.Fig. 5. Liver nucleus of a rat fed 3'-Me-DAB plus N2 diet. The nucleolus is comparatively large and round. However, a light nucleolar cap (LC) is

seen at the right corner of the nucleolus.Fig. 6. Liver nucleus of a rat fed N2 diet. The nucleolus is hypertrophie and the structure of nucleolonema is loose. The areas of pars amorpha (PA)

are well extended, and the nucleolus-associated chromatin (NAC) is thin.





AUGUST 1974 1849



M. Akao et al.

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1974;34:1843-1850. Cancer Res Mitsutaro Akao, Keiko Kuroda, Yoshihiro Tsutsui, et al. and RNA Polymerase Activity of Liver Cell Nuclei in Rats-Methyl-4-dimethylaminoazobenzene in Hepatocarcinogenesis

′Effect of Nitrofurans Antagonistic to 3

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effect of nitrofurans antagonistic to 3'-methyl-4 ......polymerase of liver nuclei (chart 1)....

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