Z. Krebsforsch. 89, 2 0 1 4 1 4 (1977)

Zeitschrift~r Krebsforschung und Klinische Onkologie 9 Springer-Verlag 1977

Cytological and Cytophotometric Studies on DMBA induced Changes of the Conjunctival Epithelium in Syrian Golden Hamsters D. Haag, F. Schlieter, V, Ehemann, and K1. Goerttler* Universit~it Heidelberg, Institut fiir vergleichende und experimentelle Pathologie (Direktor: Prof. Dr. K1. Goerttler), D-6900 Heidelberg Universit~it Bonn, Universit~its-Augenklinik (Direktor: Prof. Dr. W. Best), D-5300 Bonn, Federal Republic of Germany

Summary. A 1% solution of dimethylbenzanthracence (DMBA) in paraffinum liquidum was dropped daily into the conjunctival sac of syrian golden hamsters during 5 months. Smears of scraped conjunctival and corneal cells were obtained weekly and stained with the Papanicolaou and Feulgen techniques. The cytological alterations in the course of DMBA application were studied by morphological criteria as well as by cytophotometric measurements of the Feulgen DNA-content and the nuclear size. In controls, predominantely superficial cells with pycnotic nuclei and DNA values below the diploid DNA- content were present. The administration of DMBA was followed by inflammatory changes with a maximum on the l lth day. Attendant upon inflammation, a nuclear edema, characterized by an almost twofold enlargement of the epithelial nuclei with dispersed pale staining chromatin was observed. The nuclear edema decreased corresponding to the disappearance of the acute inflammation within one month and was followed by a second definite enlargement during further progress of carcinogen exposure. Although cells with cytological criteria of malignancy were present in the smears after two months, neither carcinoma nor carcinoma in situ could be detected histologically. The mean chromatin density of epithelial cells changed in reverse proportion to the nuclear volumes concomitant with the development of inflammation, and remained almost constant in the further course of treatment. The occurrence of cells with malignant changes was characterized by nuclear DNA values exceeding the diploid and even tetraploid range in the smears. A positive correlation between nuclear volume and DNA content in controls, established by regression analysis, was found to disappear with the incidence of DMBA-effects. The results confirm the occurrence of increased nuclear DNA-content in malignant processes and the direct action of DMBA on the level of nuclear chromatin also in the conjunctival epithelium. The changes of nuclear DNA content in exfoliated cells reflect the disturbance of differentiation in the structure of the conjunctival epithelium caused by the chemical carcinogen. Offprint requests to: Prof. Dr. Kl.Goerttler (address see above)

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Key words: 7,12-Dimethyl-benzanthracene, conjunctival epithelium, cytological alterations, cytophotometric measurements, nuclear size, nuclear DNA content, proliferation, cell differentiation, regulatory system, malignant cells.

Metaplasia, dyscariosis and carcinoma of the conjunctiva are rarities among the ocular lesions of man and laboratory animals. This suggests that a protecting mechanism against malignant transformations could exist in the conjunctival epithelium. In the literature surveyed so far, no experimental investigations on the chemical carcinogenesis of the conjunctiva were found. In the present study therefore an attempt was made to induce epithelial alterations of the conjunctiva of syrian golden hamsters by the polycyclic hydrocarbon 7,12-dimethyl-benzanthracene (DMBA), which is assumed to bind covalently to the informational molecules such as deoxyribonucleoproteins (DNP)(Brookes, 1966; Brookes, and Lawley, 1964; Jaquir and Daudel, 1964). DMBA is among the most potent skin carcinogens (Badger, 1954) and was chosen for this experiment because of its missing organotropic specifity. It is still uncertain whether a metabolic activation of DMBA is necessary for the carcinogenic activity (Arcos and Argus, 1968; Boyland, 1969), but the studies of Miller (1970, 1973) indicate, that electrophilic alkylating or arylating agents are the ultimate carcinogenic forms. For the observation of chemically induced alterations of conjunctival epithelial cells during longer periods of time, the cytologic examination of scrapings is the appropriate method. It has been established by Thygeson (1940) and was applied and worked out for the diagnosis of ocular disease by many other authors (Duszynski, 1954; Kimura and Thygeson, 1955; Gaipa, 1956; Coutifari and Nicolaou, 1959; Liolet and Iris, 1963a, 1963b; Naib et al., 1967; Dykstra and Dykstra, 1969; Naib, 1972). In these studies the Giemsa stain as well as the Papanicolaou technique had been employed for the preparation of smeared scrapings on microscope slides. On the other hand, only qualitative results emerge from the cytomorphological observations. DMBA is known to influence the template activity of chromatin, probably by reaction of free radicals with DNA (Maher et al., 1971). Therefore it was reasonable to investigate quantitative parameters such as nuclear size, chromatin concentration and nuclear DNA-content of the exposed conjunctival cells. For this purpose the Feulgen-technique combined with cytophotometric determinations by scanning measurements seemed to be suited best, since the nuclear size and the DNA value of individual Feulgen-stained nuclei is recorded simultaneously (Schiemer, 1967). Thus correlations between nuclear size and DNA content can be calculated by regression analysis and give quantitative measures of cytological changes during the experiment. Material and Methods a) Animals and Carcinogen Application 30 female inbred Syrian golden hamsters (delivered by Deutsche Versuchtstierzucht, Hannover) in the age of 2 months served as laboratory animals. Daily at the same time, one drop of a 1% solution of 7,12-Dimethyl-benz-(a)-anthracene (DMBA) in paraffin oil was administered into the conjunctival sac

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of the right eyes of 20 animals. For control, 10 animals were treated in the same manner with pure paraffin oil and kept separately from the DMBA animals. This treatment was continued for 6 months.

b) Preparationand Stainino of Smears Cytological smears were prepared weekly by careful scraping ot the conjunctivae with a stainless steel spatula. The detached material was smeared onto two slides each, which were fixed for l0 minutes immediately after smearing in pure methanol. Until staining, smears were stored in slide boxes. All slides of the experiment were stained simultaneously to prevent variations. One set of slides was stained by the usual Papanicolaou technique, another set by the Feulgen-method for nuclear DNA. The acid hydrolysis was performed in 5n hydrochloric acid at 2U C for 30 rain. After 90min of staining in Schiff's reagent (prepared according to Graumann, 1953), slides were rinsed in 3 changes of SO2-solution, 5 rain each, followed by a 10 rain rinse in tap water. The vacuum dried slides were embedded with Eukitt.

c) CytophotometricMeasurementsand Ez~aluation Cytophotometric measurements of the Feulgen-stained nuclei were performed with the scanning Universal Microspectrophotometer (UMSP 1, Carl Zeiss) which recorded simultaneously the total absorbance at monochromatic light of a wavelenght of 560 nm and the scanned area (F) of individual cell nuclei. From these data the nuclear DNA content (M) and the nuclear volume (1/) were calculated according to the formulae

M= 1/e' ~ Ei and

V = c F 3/2

i=1

where n

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E~= the individual absobances of the scanned area, n = the number of scanned points, c = a constant for the spheric shape correction. For calibration of the measurements, smears of human blood lymphocytes were processed together with the slides through the Feulgen-procedure. From their known nuclear DNA-content of 6.8x 10-12g (Metais and Mandel 1950) the specific decadic extinction coefficient=e' was calculated at 2.2 x 104 c m 2 g - 1. In the present study the conventional arbitrary units (A.U.) are divided by this constant, thus yielding the results in 10-12g. The relative accuracy of the measurements is not influenced by the division. For each point of the time schedule, at least 300 randomly selected cells were measured. To detect correlations between nuclear volumes and Feulgen-DNA values, multiple linear regression analysis of paired measurements was calculated by means of a computer program, based on Gauss' principle of least squares. The existence and significance of correlations was determined by the resulting equation and correlation coefficient/r).

Results a) Morphological Changes During D M B A Exposure U n d e r n o r m a l c o n d i t i o n s t h e s c r a p i n g s o f t h e c o n t r o l s c o n t a i n o n l y v e r y few cells f r o m t h e o u t e r s u r f a c e o f t h e c o n j u n c t i v a . T h e s e cells h a v e s m a l l r o u n d o r o v a l n u c l e i w i t h c o n d e n s e d c h r o m a t i n a n d a b u n d a n t c l e a r c y t o p l a s m (Fig. 1 a). B a s a l cells c o u l d n o t b e i d e n t i f i e d in s m e a r s o f c o n t r o l s . M o r e o r less cells w i t h c y t o p l a s m i c i n c l u s i o n s o f p i g m e n t g r a n u l a w e r e p r e s e n t in all c o n t r o l s . T h e n u c l e a r size a n d s h a p e as well a s t h e c h r o m a t i n d e n s i t y e x h i b i t o n l y s l i g h t v a r i a t i o n .

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The changes following the administration of DMBA are demonstrated in the series of pictures Fig. 1 b--h. On the l l t h day after the start of exposure the smears are rich in inflammatory cells (Fig. 1 b) and epithelial cells with enlarged nuclei which stained only pale in the Feulgen-reaction (Fig. 1 c). These cells might be ranked among the intermediary layers; isolated cells with increased nucleus/ cytoplasmic (n/c)-ratio originate from the basal cell layer. On the whole, the smears of this time represent the phenomena of acute inflammation and hyperplasia. After 4 weeks of DMBA-application the cell composition seems to be reconstituted nearly to the normal state (Fig. 1 d). Nevertheless it is more rich in cells than the controls. Differentiated epithelial cells of the cornea and conjunctiva with large nuclei, homogeneously staining chromatin, slight variation of nuclear size and abundant cytoplasm as well as a few basal cells are observed. Signs of malignant transformation are missing. After 2 month of exposure the smears are rich in cells with enlarged, homogeneously pale staining nuclei. In contrast to smears of 4 weeks some of the cells exhibit nuclear polymorphism as an indication of degenerative changes with signs of cell decomposition (Fig. 1 e). These cells might have been impaired during the inflammatory phase and are rejected in the regenerative process. They still have abundant clear cytoplasm and lack of criteria of malignancy. In the further course of D M B A administration the number of cells with indications of malignancy, such as tadpole cells (Fig. 1 I) increases from the second to the fifth month. Moreover, inflammatory cells reappear in the smears (Fig. 1 g). Compared to controls the smears of DMBA treated animals always are rich in degenerating epithelial cells (Fig. 1 h) with enlarged, hyperchromatic nuclei and poor cytoplasmic differentiation. Histologically no invasive carcinomas were observed until the 6th month. However metaplastic diturbances in the structure of the epithelium were obvious. Only one of the 20 treated animals had developed a carcinoma in situ of the cornea after 5 month. On the other hand, in all animals carcinomas of contaminated organs such as eye lids, oesophagus, pro-stomch and stomach were found. Since most of the animals died from this carcinomas between the 6th and 12th month of DMBA exposure, it was not possible to continue the observations until the development of conjunctival carcinomas.

b) CytophotometricResults The typical changes of cytophotometric results presented in this study follow the same time schedule as the morphological observations, i.e.: 11 days, 4 weeks, 8

Fig. la--h. Cytologicalchangesofthe conjunctivalepitheliumduring DMBA exposure.All magnifications x. a Control, Papanicolaou.b 11 days. Acute inflammation, Papanicolaou. e 11 days. Enlarged, pale staining nuclei. Feulgen--reaktion. d 4 weeks. Differentiatedintermediary cells with enlarged nuclei, Papanicolaou. e 2 months. Degenerativechanges, enlarged nuclei, Papanicolaou. f 4 months. Tadpole cells, Papanicolaou. g 5 months. Leucocytes and altered epithelial cells with large nuclei, Papanicolaou. h 5 month. Poorly differentiatedcells with criteria of malignancy, Papanicolaou

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weeks, 16 weeks and 20 weeks. For each point of time at least 300 measurements of randomly chosen cell nuclei of 3 different slides were evaluated. A survey of the results is given in Figures 2 and 4. From this graphs, representing the synopsis of 18 histograms, the course of changes and their correlations become evident better than in histograms. The standard deviations are designated in the graphs. Detailed results of the measurements are given completely in the Figures 3 a-f. 1. Changes of the Chromatin Density The staining intensity of the cell nuclei reflects the chromatin concentration, which is measured as the "mean absorbance". During the DMBA exposure only slight variations, following exactely in a reversed proportion the changes of nuclear size, were observed. Except the minimum concentration of 2.5% on the 1lth day, these variations were not significant (p <0.05). In the further course of exposure, the average chromatin density remained nearly constant (Fig. 2 a). 2. Changes of the Nuclear Volumes The nuclear volumes of the control smears, calculated from the scanned area of Feulgen-stained nuclei, were constant at 140 g3 within a standard deviation of +30%. On the l lth day of DMBA exposure the nuclear volumes reached a maximum of 280 g3, i.e. a twofold enlargement in the acute inflammatory phase within a standard deviation of _+30%. After fading of inflammation the mean nuclear volumes remained increased significantely (p <0.01) at values of about 200 i.t3. The variation of nuclear volumes increased correspondingly up to + 40% (Fig. 2 b). 3. Changes of the Nuclear DNA Contents The mean nuclear DNA content of scraped conjunctival epithelial cells was found to be 15% below the diploid range in controls with values of 5.8 x 10-12 grams and a variation of _+20%, only 3% of the cells had DNA values between the diploid and tetraploid range and were classified as DNA snythesizing cells due to their more dense chromatin structure. During the inflammatory phase on the 1lth day the average DNA content of the scraped cells was significantely increased by 20% (p <0.01). A proportion of 12% of the cells had tetraploid DNA values, but cells exceeding this limit (13.5 x 10-12 g) were not present in the smears. After fading away of inflammation, DNA values of the DMBA treated conjunctivae did no longer differ from controls in the 14th week. After 2 months of DMBA treatment a significantely increasing number of cells with DNA values between diploid and teraploid and even exceeding tetraploid values appeared in the smears, thus causing an increase of the average DNA content and variation of DNA values (Fig. 2 c). c) Correlations Between Nuclear Volume and DNA Content A more detailed information about the quantitative changes of cell nuclei during DMBA exposure is given in the graphs of Table 3a-f. In these graphs the nuclear DNA contents are plotted against the corresponding nuclear sizes in la3. For

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comparison, the range of controls is designated as an ellipse in all graphs. The graphs demonstrate the total of measurements, upon which the present study is based. Each point represents the related pair of DNA content and nuclear volume of one nucleus. The aggregate figure of points of the control form a uniform, coherent population. A dependence of the DNA content on the nuclear size becomes evident and is significant (p < 0.05) due to a correlation coefficient of r = 0.6. In particular the DNA content of the shrinked nuclei of superficial cells is markedly reduced compared to more intermediary cells and thus contributes to the significance of the correlation. In contrast obviously a second group of values appears in the smears of DMBA treated animals, characterized by increased and largely variable DNA contents. In the course of DMBA exposure, two different effects can be observed in the graphs: 1. Corresponding increase of nuclear volumes and DNA contents among the group of cells in the diploid DNA range, i.e. morphologically normal or stimulated epithelial cells without criteria of malignancy. 2. Occurence of an increasing group of cells with DNA values exceeding the diploid and tetraploid range with increasing time of DMBA exposure. This group of measurements results from cells with beginning or definite signs of malignant transformation and contribute to the increase of the average DNA content. These values exhibit large variations. A positive significant correlation exists between nuclear size and the DNA values (p < 0.05) if the whole of measurements is included into the regression analysis, but separated calculations for the two different types of ceils of DMBA treated animals yield no significance in the group of epithelial cells (defined by an upper limit of 9.0 x 10-12 g) and emphasize the independence of DNA content on

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the nuclear size in morphologically normal or stimulated conjunctival cells after exposure to DMBA. d) Proportion of Cells with DNA Values Above the Diploid Range The limit of diploid DNA values should be determined at 8.0 x 10-12 g. Exceeding values are assumed to result either from DNA synthesizing or hyperdiploid cells. The number of those cells is plotted against the time of DMBA exposure in Figure 4 (standard deviations of 3 slides for each point of time). During the phase of acute inflammation, the normal proportion of hyperdiploid values of about 6% was exceeded by a peak of 14% which decreased down to the normal value after 4 weeks, and was followed by a steady increase up to 42% until the 5th month.

Discussion

In the present study the attempt was made to compare the cytomorphological changes induced by the carcinogen DMBA in conjunctival epithelium with simple quantitative parameters. Those are the nuclear size, the chromatin density and the DNA content of individual cell nuclei, which were recorded cytophotometrically in Feulgen-stained smears. The cytophotometric techniques had already been employed by Rohrbach et al. (1968) to investigate the proliferative changes in the mouse skin after cocarcinogenic and carcinogenic irritations. The cytological changes during the DMBA administration are characterized by a reversible initial reactive inflammation and hyperplasia which dissapeares nearly completely after 4 weeks. This unspecific effect agrees with observations in similar experiments on the carcinogenesis of the mouse epidermis (Evensen, 1962; Iversen, 1963; Elgjo, 1966) and supports the assumption that a proliferative stimulation of the basal cell layer is caused by toxic damage and following loss of differentiated cells.The exfoliation of impaired cells could thus be one reason for the numerous scraped cells after DMBA administration. The lack of differentiated superficial cells with small pycnotic nuclei and the appearance of more intermediary and basal cells (Fig. 1c and d) is a characteristic feature of the DMBA effect. The marked increase of the nuclear volumes of the epithelial cells on the 11 th day could be interpreted as nuclear edema, according to Benninghoff (1950) attending to the reactive inflammation. Possibly the toxic effect of DMBA causes accelerated rejection of cells which could prevent complete differentiation and formation of a regularly structured epithelium. A decrease of differentiation by DMBA in vitro had already been reported by Fusenig et al. (1973) and supports this assumption. Moreover a decreased cohesiveness as an attendant symptom of disturbed differentiation could be another reason for the increase of the cell number in the scrapings. In general the present results confirm the effect of DMBA on the cell differentiation after a preceeding reversible inflammation and hyperplasia. The continued administration finally leads to dysplastic phenomena with increasing occurence of immature epithelial cells and malignant changes. Thus DMBA takes similar effects on the conjunctival epithelium as on the epidermis, which are known in many details (Iversen and Evensen, 1962).

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Obviously malignant transformations such as tadpole forms, undifferentiated cells with enlarged hyperchromatic nuclei and increased n/c-ratio appeared not before the later period of DMBA application, although the histological examinations on the end of the experiments yielded no carcinomas nor carcinoma in situ, except in one animal. Thus, morphologically two different effects can be considered as a consequence of DMBA exposure: 1. A non-specific stimulation of epithelial regeneration after toxic effects of DMBA. 2. The appearance of increasing numbers of cells with criteria of malignant transformations after about two month of DMBA exposure. These cytomorphological phases of alterations are reflected in principle also by the quantitative results of this study (Figs. 2~4). In controls, more than 50% of the nuclei had DNA values below the diploid range. In particular the DNA values of cells with pycnotic nuclei were decreased by about 40% compared to the diploid modal value of intact normal epithelial cells. This obviously reduced Feulgen-staining of chromatin in the superficial cells caused the statistically significant correlation between nuclear size and DNA values in the controls. Due to the disappearance of differentiated superficial cells after administration of DMBA, the positive correlation also disappeared among the cells in the diploid DNA range. Nevertheless a positive correlation persisted if the cells with exceeding DNA values were included into the regression analysis. Correlations between ploidy class and nuclear size have been recognized already by Jacobj (1925) and were confirmed by quantitative investigations later (Fautrez and Laquerrier, 1957; Sullivan and Garcia, 1970). The present results agree with these findings, in particular the graphs of the 11 th day and 1 month (Fig. 3 b and c) exhibit evident correlations. In correspondence with the morphological changes, two different quantitative phenomena can be distinguished: 1. Increase of the average DNA content among the poulation of exfoliated definite epithelial cells up to diploid and hyperdiploid values and the disappearance of small nuclei with reduced Feulgen-DNA values. 2. Occurence of a distinct group of cells with increased nuclear size and DNA values beyond the diploid and even tetraploid range not before 2 months of DMBA exposure. According to Rohrbach (1975) the non-specific changes are interpreted as a stimulation of proliferation due to toxic damage and loss of differentiated cells which produce proliferation inhibitory factors. The cytotoxic effect of DMBA becomes evident by the presence of a large number of cells with tetraploid DNA values on the 11 th day (Fig. 3 b). The occurence of those cells is interpreted as an impairment during the late G1- or DNA synthesis phase. Possible these cells do not undergo mitoses and are rejected in premitotic or G2-phase. The loss of cells due to the toxic effects might be compensated by an increased proliferation of the basal cells. The existence of a regulatory system of the proliferative activity is supported by the observed hyperplasia following the toxic phase (Evensen, 1961; Laerum, 1969; Iversen, 1971). Those excessive waves are known in kybernetics as a typical feature of regulatory systems with feedback. According to a model of growth regulation by Iversen (1969) the increased proportion of cells obviously in DNA

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synthesis phase on the 11 th day (peaks in Figs. 2 and 4) could well be interpreted as a regulatorY vibration of such a system, reacting on the initial toxic upset by DMBA. After continued exposure to DMBA the regulatory system might be adapted to the condition of DMBA effects by a changed steady state. It was the purpose of the present study to demonstrate the quantitative equivalents of morphological alterations during chemical carcinogenesis. The simultaneous determination of nucelar size and DNA content of Feulgen-stained nuclei proved to be a useful tool since it enables to differentiate the functional nuclear edema (Benninghoff, 1950) from malignant changes. The occurence of malignant changes in the smears runs parallel to a large degree with the occurence of cells with increased nuclear size and DNA values. Moreover those cells are identical with cell~s exhibiting the cytological criteria of malignant transformation. The present results demonstrate the locally active effect of DMBA on the conjunctival epithelium if the diluting action of the tear fluid is compensated by repeated application. Although no carcinomas of the conjunctiva had been generated during the observation period, the changes of the nuclear chromatin could be recognized even in the initial phase by means of cytophotometric measurements. These changes might be signs of initiation, similar to the initiation of the mouse skin which has been studied earlier (survey of Literature see Hecker, 1975). Concerning the chemical carcinogenesis, the conjunctival epithelium had not yet been investigated so far.

References Arcos,J. C., Argus, M. F. : Molecular geometry and carcinogenic activity of aromatic compounds. Advanc. Cancer Res. 11, 3 0 5 4 7 1 (1968) Badger, G.M.: Chemical constitution and carcinogenic activity. Advanc. Cancerr Res. 2, 73--127 (1954) Benninghoff, A.: Funktionelle Kernschwellung und Kernschrumpfung. Anat. Nachr. 1, 50 52 (1950/

51) Boyland, E.: The biochemistry of aromatic hydrocarbons, amines and urethane. In: Bergmann, E. D., Pull man, B. (Eds.): Physico-chemical Mechanisms of Carcinogenesis. The Jerusalem Symposia on Quantum Chemistry and Biochemistry, Vol. 1, pp. 2 5 4 4 . Jerusalem: Israel Academy of Arts and Sciences 1969 Brookes,P.: Quantitative aspects of the reaction of some carcinogens with nucleic acids and the possible significance of such reactions in the process of carcinogenesis. Cancer Res. 26, 1994 2003 (1966) Brookes, P., Lawley, P.D.: Evidence for the binding of polynuclear aromatic hydrocarbons to the nucleic acids of mouse skin: relation between carcinogenic power of hydrocarbons and their binding to deoxyribonucleic acid. Nature (Lond.) 202, 781 784 (1964) Coutifari, N., Nicolaou, D.: Le cyto-diagnostic en Opbtalmologie. Arch. Ophthal. (Paris) 19, 360 (1959) Duszynski, L. R. : Cytology of conjunctival sac. Amer. J. Ophthal. 37, 576--578 (1954) Dykstra, P., Dykstra, P.A.: The cytologic diagnosis of carcinoma and related lesions of the ocular conjunctiva and cornea. Trans. Amer. Acad. Ophthal. Otolaryng. 73, 979--995 (1969) Elgjo, K.: Epidermal cell population kinetics in chemically induced hyperplasia. Oslo: Universitetsforlaget 1966 Evensen, A. : Changes in the synthesis of deoxyribonucleic acid (DNA) and in mitotic count in epidermis of hairless mice after a single application of 1 percent 3-methylcholanthrene in benzene. Acta path. microbiol, scand Suppl. 148,43 52 (1961) Evensen,A.: Effects of carcinogens on cell proliferation. In: Iversen, O.H. and Evenson, A.: Experimental skin carcinogenesis in mice. Acta path. microbiol, scand. Suppl. 156, 93 184 (1962)

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