4-Nitroquinoline N-oxide is a derivative ofquinoline which was first described by Ochiai in1943 (11). Since that time this carcinogenic agenthas been extensively studied, and Nakahara hasrecently reviewed the subject (9). One of the biologic effects described was the production of ribonucleoprotein(RNP)-containing intranuclear inclusions or vacuoles (4, 5). This change was observed in Chang liver cells which were treated witha 10 M concentration of 4-nitroquinoline N-oxideand subsequently stained with hematoxylin andeosin (Figs. 4, 5). The purpose of this paper is todescribe additional cytologic changes in Changliver cells treated with 4-nitroquinoline N-oxide.

MATERIALS AND 1@IETHODS

Chang liver cells maintained in Eagle's basicmedium and Hanks balanced salt solution wereutilized for all studies reported in this paper. Forexperimental purposes the cells were grown intissue culture chambers and on 1-inch round coverslips in ointment jars (approximately 50,000cells/cc culture Inedium). The experiments wereperformed by removing the medium and replacingit with a 10@ M solution of 4-nitroquinoline N-oxide' in Hanks balanced salt solution. (Other

* This research was supported by Tobacco Industries Re

search Grant No. 8778 and Damon Runyan Fund No. 8773.

1 4-Nitroquinoline N-oxide provided through the generosity

of Dr. Hideya Endo, Cancer Institute, Japanese Foundationfor Cancer Research, Tokyo.

Received for publication September 6, 196%.

corn,entrations of 4-nitroquinoline N-oxide varyingfrom 10@ to 10@ M, with periods of exposure varying from 10 minutes to 6 hours, have also beenutilized. The best demonstration of the nucleolarchanges to be described subsequently has beenachieved with the 10@ M solution of 4-nitroquinoline N-oxide and a 10-minute exposure of the cellsto the chemical.) After 10 minutes the 4-nitroquinoline N-oxide was removed and replaced byculture media. Phase-contrast microscopic imagesof the cells were then recorded by time-lapsecinematography. At intervals of 2, 4, 6, 12, and 24hours coverslip preparations were removed fromthe ointment jars and fixed for electron microscopyand for various histological stains. Dalton's solution and 2 per cent potassium permanganate indistilled water (8) were used as fixatives for deetron microscopy. Following fixation the cells weredehydrated in graded alcohols and embedded inmetliacrylate. For light microscopic studies thecoverslip preparations were fixed in Carnoy's solution and stained with hematoxylin and eosin,azure “B,―and acridine-orange (1).

RESULTS

In living cells the principal morphologic effectsof 4-nitroquinoline N-oxide are seen in the nuclei.Cytoplasmic alterations, although present, are lessstriking and appear later. The sequential changesproduced by 4-nitroquinoline N-oxide in Changliver cells in vitro are described below.

Nucleolar exhaustion .—Within 2 hours after

535

Nucleolar “Caps―—aMorphologic Entity Produced bythe Carcinogen 4-Nitroquinoline N@Oxide*

ROLLAND C. REYNOLDS, PHILIP O'B. MONTGOMERY, AND DAVID H. KARNEY

(Department of Pathology, University of Texas Southwestern Medical School, Dallas, Texas)

SUMMARY

Distinctive morphologic changes produced by 4-nitroquinoline N-oxide in Changliver cells have been demonstrated by means of histologic stains, electron microscopy,and time-lapse motion picture studies of the phase-contrast microscopic images ofliving cells. The principal changes include : (a) nucleolar exhaustion manifested by aprogressive decrease in the size of the nucleoli, (b) fusion of the nucleoli, and (c) separation of the pars amorpha and the nucleoloneme to produce two types of nucleolar“caps.―The possible relationships between these morphologic changes and the knownbiochemical activities of 4-nitroquinoline N-oxide are discussed.

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536 Cancer Research \T01 23, May 1963

treatment of the culture there was a marked decrease in the size of the nucleoli. This was demonstrated best by the azure “B―stain (Figs. 1, 2,and 3). With the decrease in size there was a concomitant change in the shape of the nucleoli consisting of a loss of the irregular contour whichcharacterizes the nucleoli of untreated cells, andthe development of a spherical shape (Figs. 1, 2,and 8). As the nucleoli changed in size and shapethey occasionally fused. This could be clearlydemonstrated by time-lapse motion pictures withphase-contrast microscopy. It appears probablethat the nucleolar fusion observed in this studywas due to the effects of 4-nitroquinoline N-oxide,since this alteration did not appear in untreatedcells.

Formation of nucleolar caps.—The most strikingchange demonstrated in the treated cells wasa redistribution of the morphologic components of the nucleolus. In untreated cells fixed inDalton's solution and studied with electron microscopy the nucleolus consisted of strands orcords of electron-dense particles, the nucleoloneme,and a more electron-transparent portion, the parsamorpha (Fig. 6). Occasionally a slight margination of the nucleoloneme produced a condensationof electron-dense particles at the edge of thenucleolus. The various morphologic patterns seenin the nucleoli of untreated cells in our materialresembled those described by Davis (2) in nucleoliof normal rat liver cells.

In cells treated with 4-nitroquinoline N-oxide adistinct change occurred in the distribution of thepars amorpha and the nucleoloneme, as early as3—4hours after the introduction of the agent. Thisredistribution could be visualized with phase microscopy and with electron mici@oscopy. With deetron microscopy dark nucleolar “caps―of marginated electron-dense particles were readily seen intreated cells fixed with Dalton's solution (Figs. 7,8, and 9). These nucleolar “caps―had the sameelectron density as the darkest particles of thenucleoloneme. In some instances only these dark“caps―were present (Figs. 8, 9). In others (Fig. 7)there was the dark nucleolar “cap,―and, in addition, a second bulging nucleolar mass was presenton one side of the nucleolus. This latter structureproduced a “bubble-like―mass of material whichwas composed of less electron-dense particles thanin the central area of the nucleolus. These structures have been designated light nucleolar “caps.―One of thesetwo patternsof redistributionofnucleolar particles was found in over 50 per centof cells surviving treatment with 4-nitroquinolineN-oxide for longer than 4 hours.

Fixation of the cells with 2 per cent potassium

permanganate in distilled water resulted in a somewhat different appearance of these nucleolarchanges. In the untreated cell fixed with potassium permanganate the borders of the nucleoliwere not distinct, and there was an intimate association of the heterochromatin and the electrondense particles of the nucleolus (Fig. 10). Fourhours following treatment the edges of the nucleolibecame sharply outlined and clearly showed margination of electron-dense particles, presumablyderived from the nucleoloneme (Figs. 11—13).Inaddition, a sharply demarcated oval mass wasoften seen at one side of the nucleolus; it resembledthe surrounding nucleoplasm except for its consistent relationship to the nucleolus. This structureis also called the light nucleolar “cap.―

In the living cell phase-contrast microscopy revealed the same nucleolar changes observed withthe electron microscope (Figs. 18, 19, 21—23).Astudy of time-lapse motion picture films coveringa period of 4 hours showed that some of the clearareas of the pars amorpha were lost, while othersgradually coalesced and migrated to one side ofthe nucleolus (Fig. 18). As time progressed, material from the nucleoloneme continued to migrate to one side of the nucleolus, while the parsamorpha collected as nipple-like “caps―along thenucleolar margin.

Nuclear changes.—Figure 4 demonstrates theH. & E. appearance of the nuclear inclusions produced by 4-nitroquinoline N-oxide (4, 5). FigureSis a photomicrograph of these nuclear changes asseen with the fluorescent microscope. With fluorescence microscopy after acridine-orange staining,the nuclear inclusions appeared as vacuoles whichcontained a small amount of red fluorescing material compatible with RNP. The morphology ofthese inclusions or vacuoles when studied withelectron microscopy varied with the type of fixative.They could not be demonstrated after osmic acidfixation. After fixation with 2 per cent potassiumpermanganate they were seen as round bodies inthe nuclçoplasm. Figure 10 is an electron photomicrograph of an untreated Chang liver cell fixedin 2 per cent potassium permanganate. There wasan irregular distribution of the electron-denseparticles of the heterochromatin around the nucleolus and along the nuclear membrane. In thenucleoplasm the pattern of the heterochromatinwas suggestive of irregular cords. In treated cellsthe heterochromatin appeared to condense in acircular pattern about areas of the nucleoplasm,producing a vacuolated appearance in the nucleussimilar to that seen with the fluorescencq@ microscope (Fig. 11).

Cytoplasmic changes.—In addition to nuclear

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REYNOLDS et al.—Nucleolar “Caps―—a Morphologic Entity 537

changes other morphologic alterations were foundin cells treated with 4-nitroquinoline N-oxide.Four to 6 hours after treatment there was a detectable loss of cytoplasmic basophilia which couldbe demonstrated with the azure “B―stain. Inacridine-orange stains the loss of pink fluorescence,presumably owing to RNP, was not as striking aswas the loss of cytoplasmic basophilia with theazure “B―stain. Electron micrographs demonstrated that the endoplasmic reticulum of the cytoplasm remained intact (Figs. 11, 12).

Effect on dividing cells.—4-Nitroquinoline N-oxide had a profound effect on cells undergoingmitosis. The first evidence of cellular damage dueto 4-nitroquinoline N-oxide was seen in dividingcells. It was unusual to find an intact mitotic figure2 hours after the cells had been exposed to a 10@ Mconcentration of 4-nitroquinoline N-oxide. A moredetailed study of the effects of 4-nitroquinolineN-oxide on the dividing cell is in progress andwill form the basis of a subsequent report.

DISCUSSION4-Nitroquinoline N-oxide is a simple chemical

compound in which the specific moieties responsible for its carcinogenic activity have been elucidated. Nakahara, Fukuoka, and Sakai (10) havedetermined that the nitro group at position 4 andthe oxygen at position 1 are both necessary for theproduction of skin carcinomas in mice. Derivativesof 4-nitroquinoline N-oxide with ethyl, methyl, orchlorine groups at positions 2 and 6 retain the carcinogenic activity of the parent compounds. Theelectron configuration of 4-nitroquinoline N-oxideis such that the nitro group at position 4 is highlyreactive and undergoes rapid substitution reactions with nucleophilic compounds, particularlythose containing 511 groups (6). Okabayashi (12)and Endo (3) have both demonstrated that sulfhydryl groups are highly reactive with 4-nitroquinoline N-oxide. No reaction was demonstratedbetween 4-nitroquinoline N-oxide and carboxyl oramino groups.

Other biochemical effects of 4-nitroquinolineN-oxide on cellular metabolism have been described by Fukuoka and co-workers (7, 13). In animportant series of experiments utilizing cancercells in vitro these workers demonstrated thattreatment of these cells with 4-nitroquinoline N-oxide resulted in the following alterations in cellular metabolism: (a) an early arrest in glycolysispresumably owing to the inhibition of the SHcontaining enzyme triosephosphate dehydrogenase and a decrease in cellular DPN, (b) a markeddecrease in cellular content of adenosine triphosphatase (ATP) resulting from the inhibition of

glycolysis and subsequent failure of regenerationof ATP, (c) a decreased uptake of @32into nucleicacids and of C'4-labeled amino acids into protein.Fukuoka attributed these changes to the loss ofATP, since the uptake of @32and C'4-labeled aminoacids was restored in microsomal fractions of 4-nitroquinoline N-oxide-treated cells to which ATPwas added. In the opinion of Fukuoka and coworkers, the major biochemical alterations incellular metabolism produced by 4-nitroquinolineN-oxide resultfrom inhibitionofglycolysis, throughinactivation of SH-containing enzymes and depression of cellular DPN levels, resulting in adecreased ATP content in the cell.

4-Nitroquinoline N-oxide is unique in being acarcinogenic agent which produces characteristicnuclear alterations. These alterations consist ofthe nuclear RNP-containing inclusions first described by Endo (4, 5) and the alterations innucleolar structure described in this paper. Therelationship between the morphologic changesproduced by 4-nitroquinoline N-oxide and itsother biological and biochemical activities may bespeculated upon at the present time. One possibleworking hypothesis might encompass the followingpoints : (a) As a result of decreased intracellularDPN and ATP levels, protein and nucleic acidsynthesis within the cell is inhibited (7, 13). (b)The cell responds to decreased protein synthesiswith a discharge of preformed RNP from thenucleolus to the cytoplasm in an attempt to restore adequate protein synthesis. This results innucleolar exhaustion manifested by the decreasedsize of the nucleoli, the occasional fusion ofnucleoli, and the physical redistribution of thenucleoloneme and pars amorpha to form nucleolar“caps.―The RNP-containing nuclear inclusionsmay represent masses of RNP in transit from thenucleolus to the cytoplasm. (c) Since nucleic acidsynthesis is also inhibited the available nucleolarRNP is completely exhausted, and the cell dies.

The demonstration of the nucleolar “caps―produced by 4-nitroquinoline N-oxide provided further insight into the ultrastructure of the nucleolus. In the normal Chang liver cell two distinctcomponents of the nucleolus are seen, the strandlike nucleoloneme and the vacuolated areas whichwe have referred to as the pars amorpha (Figs. 6,14, 20). In the time-lapse motion picture studies(Figs.14—19)twovacuolesofthenucleolusareseento coalesce and migrate to one side of the nucleoluswhere they form a light nucleolar “cap.―This observation would appear, therefore, to establishthe nucleolar vacuoles as distinct morphologiccomponents of the nucleolus rather than representing defects or holes in the nucleolus.

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538 Cancer Research Vol. 23, May 1963

ACKNOWLEDGMENTS

Grateful appreciation is extended to Dale McClendon,Nancy Arnold, Patsy Johnson, and Emil Sanders for their assistance in tissue culture, photography, and electron microscopy.

REFERENCES

1. BERTALANFFY,L.; MA5IN, M.; and MA8IN, F. A New andRapid Method for Diagnosis of Vaginal and Cervical Cancer by Fluorescence Microscopy. Cancer, 2:873-87, 1958.

2. DAvis, J. M. G. The Ultrastructure of the MammalianNucleolus. In: J. S. Mrrcnzu@ (ed.), Cell Nucleolus, Chapter I. New York: AcademicPress, 1960.

3. ENDO,H. On the Relation between Carcinogenic Potencyof 4-Nitroquinoline N-oxides and the Reactivity of TheirNitro-groups with S-H Compounds. Gann, 49:151-56,1958.

4. ENDO,H.; Aoxi, M.; and Aoy@att, Y. Formation of Nuclear Inclusion Bodies in Tissue Culture Cells by 4-Nitroquinoline N-oxide. Gann, 50:209—17, 1959.

5. ENDO,H.; TAKAYAMA,S. ; KA&JGA,T. ; and OHASHI,M.Histochemical Studies on Nuclear Inclusion in Tissue Culture Cells Induced by .4-Nitroquinoline N-oxide. Gann,52:173—77,1961.

6. Fuxui, K. ; IMAMurts,A. ; and NAGATA,C. Relation be

Fio. 1.—Untreated Chang liver cells stained with azure“B.―Note size and irregularity in shape of the nucleoli (N).Mag. X360.

Fia. 2.—Chang liver cells 2 hours after treatment with a10@ Msolution of4-nitroquinoline N-oxide. The nucleoli (N) arereducedinsizeand show a uniformsphericalshape.Note alsothe loss of cytoplasmic basophilia. Mag. X350.

FIG. 3.—Chang liver cells 4 hours after treatment with

4-nitroquinoline N-oxide. Nucleoli (N) show no further decrease in size. Further loss of cytoplasmic basophilia is present.

tween the Electronic Structure and Carcinogenic Activityof 4-Nitroquinoline N-oxide and Related Compounds.Ganu, 51:119—23, 1960.

7. Fuxuos@, F. ; ONO, T. ; OHASHI, M. ; and NisHnwrm@,S. Effect of 4-Nitroquinoline N-oxide on the Metabolismof Cancer Cells in vitro. Gann, 50: 1, 23—25,1959.

8. MOLLENHAUER, H. H. , and ZEBRUN, W. Permanganate

Fixation of the Golgi Complex and Other CytoplasmicStructures of Mammalian Testes. J. Biophys. Biochem.Cytol., 8:761—75, 1960.

9. NAKAHARA,W. Critique of Carcinogenic Mechanism. Prog.Exp. Tumor lIes., 2:158—202, 1961.

10. NAKAHARA,W. ; FUKUOKA, F. ; and SAKA!, S. The Relationbetween Carcinogenicity and Chemical Structure of Certhin Quinoline Derivatives. Gann, 49:33—41, 1958.

11. OCHIAI, E.; ISHIKAWA, M.; and SAL, S. Polarization ofAromatic Heterocyclic Compounds: XXX Nitration ofQuinoline N-oxide. J. Pharmaceut. Soc. Japan, 63:280—8'2,1943.

1@2.OKABATASHI,T. Studies on Antifungal Substances.VI. Onthe Reaction between 4-Nitroquinoline N-oxide and S-HCompounds. J. Pharmaceut. Soc. Japan, 73:946—50, 1953.

13. ONo, T. ; TOMARU,T.; and FUKUOKA,F. Effect of 4-Nitroquinoline N-oxide Derivatives on the DPN DependentEnzyme Systems. Gann, 50: 189—200,1959.

Mag. X350.Fm. 4.—Nuclear inclusions (NI) produced by 4-nitroquino

line N-oxide demonstrated by hematoxylin and eosin afterfixation in Carnoy's solution. Mag. X550.

FiG. 5.—Nuclear inclusions (NI) produced by 4-nitroquinoline N-oxide appear as vacuoles containing a small quantity ofpink-fluorescing material when stained with acridine-orangeand viewed with the fluorescent microscope. The pink fluorescence suggests the presence of RNA in these inclusions. Mag.x500.

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FiG. 6.—Electron micrograph of the nucleolus of an untreat

ed Chang liver cell fixed with Dalton's solution. Note theskeinlike network of the nucleoloneme. The pars amorphaappears as lighter areas between the nucleoloneme. Mag.x10,000.

Fios. 7—9.—Electron micrographs of Chang liver cells aftertreatment with 4-nitroquinoline N-oxide and fixation inDalton's solution. Distinctive morphologic changes may beseen in the nucleoli (N). The marginal condensation of electrondense particles gives rise to dark nucleolar “caps―(DNC). InFigure 7 the bulging mass of nucleolar material (LNC) isthought to correspond to the light nucleolar “caps―seen withpotassium permanganate fixation and the phase microscope(Figs.1%,13,19,and %@).Mag. X10,000.

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FIG. 10.—Untreated Chang liver cells fixed with potassiumpermanganate. The nucleolus (N) and its relationship to thesurrounding “cord-like―strands of heterochromatiti are welldemonstrated. Mag. X 10,000.

FIGS. 11—13.—Electron micrographs of Chang liver cellsfixed with potassium permanganate after treatment with4-nitroquinoline N-oxide. Early nucleolar changes are seen inFigure 11. They consist of condensation of dark particlesaround the nucleolus (DNC) and a bulging mass of light particles (LNC). Figures 1@and 13 show the later stages of thisprocess. Dark nucleolar “caps―(DNC) are seen at the marginsof the nucleoli. Both nucleoli show nipple-like “caps―(LNC)composed of particles with an electron density similar to thesurrounding nucleoplasm. These light nucleolar “caps―maybe distinguished from the nucleoplasm in permanganate-fixedmaterial oniy by their consistent relationship to the nucleoli.The similarity of these altered nucleoli may be compared withthose seen with the phase microscope (Figs. 19, ‘21—'23).Mag.X 10,000.

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FIGS. 14—19.—Sequential nucleolar changes in a livingChang liver cell treated with 4-nitroquinoline N-oxide. Thesephotographs represent single frames taken from a 16-mm.phase time-lapse motion picture covering a period of approximately 4 hours. In Figure 14 the nucleolar pattern resemblesthat seen with the electron microscope after fixation with Dalton's solution (Fig. 6). There was a progressive loss of smallerclear vacuolated areas (pars amorpha) of the nucleolus (Figs.15—17)and the progressive coalescence of the larger vacuoles.Two areas of the pars amorpha migrated to the left-hand sideof the nucleolus and formed a light nucleolar “cap―(LNC)(Figs. 18, 19@.In Figure 18 the dark material of the nucleolusforms a dark nucleolar “cap.―A section taken verticallythrough the nucleolus in Figure 19 would probably show darknucleolar “caps―similar to those seen in electron micrographs(Figs. 8, 9). Mag. X@,500.

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FIG. %0.—Phase-contrast photomicrograph of untreatedChang liver cell seen with the oil-immersion phase microscope.The nucleolus (N) contains intertwining strands of dark contrast material. Mag. X3,500.

Fios. %1—%3.—Phase-contrast photomicrographs of Changliver cells after treatment with 4-nitroquinoline N-oxide. Lightnucleolar “caps―(LNC) have formed, and the margination ofdark contrast material forms dark nucleolar “caps―(DNC).Cf. Figures 7—9,1%, and 13. Mag. X3,500.

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1963;23:535-538. Cancer Res   Rolland C. Reynolds, Philip O'B. Montgomery and David H. Karney  Carcinogen 4-Nitroquinoline N-Oxide

a Morphologic Entity Produced by the−−Nucleolar ''Caps''

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