Kinetics of induction and growth of precancerous liver-cell foci, and liver tumour formation by diethylnitrosamine in the rat

Europ. J. Cancer Vol. 1I, pp. 689-696. Pergamon Press 1975. Printed in Great Britain

Kinetics of Induction and Growth of Precancerous Liver-Cell Foci, and Liver Tumour Formation Diethylnitrosamine in the Rat* E. SCHERER and P. EMMELOT Department of Biochemistry, Antoni van Leeuwenhoek-Laboratory, The Netherlands Cancer Institute, Amsterdam, The Netherlands. Abstract--Induction of loci of atypical liver cells ~A TPase-deficient zslands, which previous& have been shown to serve as tumour ceU precursor) by a single dose of diethylnitrosamine in partial& hepatectomized rats showed direct proportionality between number of islands and dose up to 30 mg DENA/kg, pointing to a one-hit mechanism of induction. The curve relating number of islands to dose levelled off at >- 50 mg/kg, and this was interpreted as resulting main&from the toxic action of the carcinogen. Island size (the number of cells per island) was independent of dose up to 30 mg DENA/kg, but progressive& increased in the higher dose range. Increased proliferation of island cells was attributed to the regenerative stimulus stemming from the toxic effect of the carcinogen at the higher doses. Analogous findings resulted from the measurement of island induction in the livers of intact rats receiving 0"9--14 mg DENA[kg daily for 4 weeks. A single dose of >= 50 mg DENA/kg produced hepatocellular tumours in the partial& hepatectomized rat. Liver tumour formation in this system is discussed and the significance of cell proliferation (DNA synthesis) for the various phases of the hepatocarcinogenic process is stressed.

INTRODUCTION

which we use for the routine identification and quantitation of loci of these cells. Most probably [6], the foci---designated as islands--are clonally derived from liver cells acted upon by carcinogen, in our experiments mostly DENA; thus the sequence: liver cell ~ island-forming cell ~ island = population of island cells, identified as an ATPase-deficient focus surrounded by normal, ATPase-positive liver cells, is obtained. The island cells are endowed with the capacity to proliferate for a considerable period of time in the absence of further treatment in an otherwise normal and resting liver. The number of islands induced is markedly enhanced by partial hepatectomy performed 2 4 h r before the application of 10-20rag DENA/kg [5]. Islands thus induced do not attain the malignant state and only seldom

CARGINOGENESIS is a process in which tumour cells are derived from normal cells, apparently by a number ofstepwise alterations [1-4]. A cell type that may qualify as the first morphologically detectable cell stage causally involved in chemical carcinogenesis in the rat liver has recently been identified [5]. These cells are persistently changed in morphological, enzyme histochemical and growth properties from the normal parenchymal cells. ATPase deficiency is the main phenotypic marker

Accepted 22 May 1973. *Part of this investigation has been communicated to the Eleventh International Cancer Congress, Florence, 22-26 October 1974 (Abstracts, Part 3, p. 354). 689

690

E. Scherer and P. Emmelot

reach macroscopic dimensions within the life span of the rats [7]. Recent quantitative experiments [7] support the view that these island cells are causally related to tumour formation, serving as tumour cell precursors by being substrate for further carcinogenic action. Accordingly, induction of tumour cell precursors (island cells) and conversion of the latter to turnout cells can be studied separately and quantitatively, allowing dose-response relations to be established for the separate processes. The present paper reports on the doseresponse relationship of island induction by DENA in rat liver (partially hepatectomized and single application; intact and multiple application), quantitating (a) the number of islands formed, (b) the number of cells per island (from which the growth rate of the island cells may be derived), and (c) the total number of island ceils present after a fixed period of time as a function of the dose of DENA. On the basis of the finding that a single dose of > 50 mg/kg administered to partially hepatectomized rats leads to liver tumours, and taking morphological data into account, liver carcinogenesis in this system is discussed as a process manifesting itself in at least two stages, i.e. island cell induction and focal progression of some island cells to tumour cells. MATERIALS AND METHODS Female rats of the Sprague-Dawley strain (specific pathogen-free, Zentralinstitut ftir Versuchstierzucht, Hannover, Germany) were fed a standard diet (Hope Farms, Woerden, The Netherlands) and given water ad libitum. Body weight amounted to 200-300 g with a deviation of + 25 g at the start of an experiment. Two-thirds hepatectomy was performed according to Higgins and Anderson [8] between 10 a.m. and 3 p.m., 20-24 hr prior to DENA application. DENA (Merck-Schuchardt, Mfinchen, Germany) in tap water, was applied by stomach tube in the concentrations indicated below. The daily doses were administered 6 days per week in an amount of 7/6 the dose indicated in the text. For quantitation of the islands, rats were killed 8 weeks after induction following a 16 hr fasting period. Livers were removed, weighed, and representative portions of the anterior and posterior right, and the two caudate lobes (in the case of hepatectomized rats) and of total liver (in the case of intact rats) were frozen on solid CO 2. Cryostat sections (10pm) were stained for ATPase, glucose-6-phosphatase and PAS

reactions and by hematoxylin and eosin as described [5]. The quantitation of island number and size has been reported in detail previously [5]. Depending on island number, from 3 to 15 cm 2 of ATPase stained sections were evaluated per animal. DNA content of the liver was measured by the Schmidt-Thannhauser method [9] and the indole reaction [10] 72 hr after partial hepatectomy corresponding to 48 hr after a single application of D E N A using 4 rats per dose level. For the study of liver tumour formation rats were treated as mentioned below and were kept under standard conditions until they became moribund and were killed. Livers were weighed and pieces from tumours and each lobe were frozen on solid CO2 for histological examination of sections stained as indicated above.

RESULTS

Single dose application--regenerating liver Single doses of DENA ranging from 0.3 to 140 mg/kg were administered orally to rats 24 hr after two-thirds hepatectomy, 4-9 animals being used for each dose level in 3 experiments comprising 0.3-30, 0.6-10, and 50-140mg DENA/kg. The highest dose was lethal but provision of 10% glucose in the drinking water kept half of these rats alive. After 8 weeks ATPase-deficient islands of a similar character as described previously [5], were present in the livers of all rats. In rats which had received a dose of up to 70 mg DENA/kg, liver tissue surrounding the islands was normal in respect to structure, cell morphology, ATPase and glucose-6-phosphatase staining. Higher doses may lead to bile-filled cysts and cirrhosis. Effect of DENA on liver regeneration The number of islands induced by a given dose of DENA is considerably enhanced by prior two-thirds hepatectomy [5]. Most probably, DNA synthesis occurring in the regenerative response, enhances the sensitivity of the liver cells to the action of the carcinogen [11-13]. Thus, for dose-response studies in partially hepatectomized rats it is essential to know if liver regeneration is affected in the dose range used. Liver weight and DNA content are recorded in Fig. 1 as a function of D E N A dose, 48 hr after the farter's application which corresponds to 72 hr after partial hepatectomy. No effects were seen up to 30 mg DENA/kg, but a dose of 50 mg/kg significantly retarded liver regeneration as shown by the decrease in liver weight and DNA content.

Kinetics o f Induction and Growth o f Precancerous Liver-CeU Foci, and Liver Tumour Formation # .

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Fig. 1. Weight and D N A content of rat liver 72 hr after partial hepateetomy, as function of the dose of a single application of DENA given 24 hr after the operation. The mean liver weight and D NA content before partial hepatectomy amounted to 9 g and 29 mg respectively. Seventy per cent of the livers were removed by the operation.

691

DENA/kg. At higher doses (50-140 mg/kg) the size of the islands increased progressively with dose. Since the mean size of the cells in the various islands was similar and independent of dose, the number of cells per average island (calculated from the mean island volume) was similar up to 30 mg DENA/kg, but increased from 50 to 140 mg/kg (Table 1). Since islands are most probably derived from single liver cells altered by DENA action [6], it follows that island cells induced by the higher DENA doses had proliferated faster than had those induced by the lower doses. Calculation (from Table 1) of the mean population doubling time of the island cells, assuming all cells to be in cycle and no cell loss, showed

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Number o f islands versus dose

The dose-response curve for island induction is illustrated in Fig. 2 in double logarithmic plot. The curve consists of an ascending limb, a plateau and a descending limb. The ascending limb has a slope of + 1 and therefore direct proportionality is obtained between number of islands induced and dose in the range of 0"3 to 30 mg DENA/kg. For doses of => 50 mg DENA/kg no further increase but rather a slight decrease in island number at the highest doses was obtained. Such a diminuation of the response may be the result of toxic manifestations (see previous section) and/or changes in the metabolism of DENA at high dose level.

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Fig. 2. Number of A TPase-deficient islands as function of the dose of a single application of DENA to partially hepatectomized rats. Results of three experiments. Vertical bars indicate the s.d., the slope of the straight line is+ 1. Island size vs. dose

As illustrated in Fig. 3 the size distribution of the islands 8 weeks after induction was about equal for islands induced by doses up to 30 mg

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Fig. 3. Size dbtdbution of A TPase ~fic~nt islands as a function of the dose of a single application of DENA to

partially hepatectomized rats. The mean error is indicated by vertical bars; the dose by figures (mg/kg).

that the cells induced by the highest dose (140rag DENA/kg) proliferated about two times faster than did island cells induced by 30 mg DENA/kg and lower doses. Increased island cell proliferation appears to come into operation at a dose level of about 50 mg DENA/kg and thus coincides with the beginning of the plateau in the dose-response curve of island induction (Fig. 2) and the manifestation of toxicity (Fig. 1). Thus, rather than being intrinsically capable of faster growth, the island cells induced by the higher DENA doses may proliferate more rapidly as a result of the toxic effect of this dose level on the liver (see "Discussion"). Total number o f island cells per liver versus dose

From the number and size of the islands and the number of cells/era 3 of island tissue [5], the volumetric fraction occupied by island cells and the total number of these cells in the livers 8 weeks after induction by the various single doses of DENA, can be calculated. Results

692

E. Scherer and P. Emmelot Table 1. Mean number of parenchymal cells per ATPase-deficient island 57 to 61 days after DENA application to 2/3 hepatectomized rats One cm s of island tissue contains 7.8 x 107 cells [5]. Dose of D E N A (mg/kg) Cells p e r island

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1"2

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10

30

50

70

100

140

144

125

100

119

125

90

190

365

1830

3700

illustrated in Fig. 4 show that the curve relating the volumetric fraction and the total number of the island ceils to the dose has two main portions: 1. A linear increase in the low dose range due to the direct proportionality between dose and number of islands of equal size and cell content. 3

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Multiple dose application--intact liver A similar type of experiment was carried out with intact rats (no partial hepatectomy, 4 rats per dose level) which received 0.9-14mg DENA/kg daily for 4 weeks, measurements being made after another 6 weeks. The liver surrounding the ATPase-deficient islands was

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number of ATPase-deficient islands and dose, the latter result implies that a similar relation exists between dose and induction of islands with the two additional characters.

Volumetric fraction of liver occupied by A TPaseislands and calculated number of island cells as of the dose of a single application of DENA to hepatectomized rats. Results from three experiments.

2. A sudden increase in the high dose range where the decrease in island number is far outweighed by the increase in number of cells per island due to their faster proliferation. Other phenotypic markers It has been demonstrated previously [5] that among islands a distinction can be made according to whether they demonstrate ATPase deficiency alone or glucose-6-phosphatase deficiency and/or glycogen retention after fasting in addition. The correlation between ATPase deficiency and the other two phenotypic characters was studied quantitatively on parallel sections for the dose range 0.6-10 mg DENA/kg. The relative frequencies of the three staining characters appeared to be independent of dose in this range. Thus for all doses examined it was found that of the ATPase-deficient islands, about 17% contained glycogen after fasting and that half of the latter islands was also deficient in glucose-6-phosphatase staining. Given the direct proportionality between

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normal for doses up to 7 mg/kg day; at 14 mg/ kg day, biliary cysts were present. The doseresponse curve in terms of number of islands induced is illustrated in Fig. 5. For the lower doses at least, a slope of + 1 is obtained, and, although a straight line could be constructed for the whole dose range, it appears that irregularities occur (see dotted lines for s.d.) above 3"5 mg/kg day, a dose which previously has been shown to exert a toxic effect in rat liver [7]. The volumetric fraction of liver occupied by island cells and the total number of these cells as a function of DENA dose shows a marked increase between 7 and 14 mg DENA/kg day

Kinetics of Induction and Growth of Precancerous Liver-Cell Foci, and Liver Tumour Formation

(Fig. 5), reflecting the increase in the size of the islands (growth rate of the island cells) induced by the latter as compared with the former dose level (Fig. 6). At =< 7 mg DENA] kg day the size distribution of the islands was

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independent of dose, and similar to that obtained after single low doses and partial hepatectomy (Fig. 3). Parenthetically, glucose-6-phosphatase deficient islands were present in small number, amounting to 5-10% of the number of ATPasedeficient islands. Some 90% of the glucose-6phosphatase deficient islands were also ATPase deficient. The islands which were both ATPase and glucose-6-phosphatase deficient were generally somewhat larger than ATPasedeficient islands positive in glucose-6-phosphatase. The very few glucose-6-phosphatase deficient islands normal in ATPase staining were small with a diameter of up to 110 #m.

Table 2.

693

Liver turnout formation by a single application o f 50 mg D E N A / k g , or higher, to partially hepatectomized rats Two to three month-old female Sprague Dawley rats used throughout this investigation, received one dose of 20, 50, 70, 140 oI 200 mg DENA/kg 24 hr after two-thirds hepatectomy. Apart from acute death, all animals had died by (sacrificed when moribund, e.g. from mammary tumours) or were sacrificed after 24 months. Confirming previous results [7], a dose of 20 mg/kg did not produce carcinomas; only many microscopically large and a few macroscopical islands were observed. However, various types of liver tumours, adequately described by other authors [14, 15], were obtained by the higher DENA doses (Table 2). The hepatocellular tumours were mainly trabecular, well differentiated, and contained glycogen. Glycogen-free carcinomas, lacking trabecular organization and cytoplasmic differentiation of basophilic material were also present next to more anaplastic tumours. The latter tumours, which were the main cause of death by leading to abdominal hemorrhage were indistinguishable from similar tumours found after repeated DENA application; it has previously been argued [7] that such tumours may be of hepatocellular origin. At > 100 mg DENA/kg, no such malignant tumours were encountered, only well differentiated glycogencontaining hepatomas being present; the reproducibility of this result should, however, be tested in a larger experiment. In 4 of 7 animals of these groups, the livers were completely altered by very large biliary cysts. In the 50 and 70 mg/kg groups the tumours were present in livers which contained welloutlined islands; disorganization of the liver structure hampered clear recognition of islands

Formation of hepatocellular carcinomas after a single dose of D E N A given to partially hepatectomized rots i

Dose mg DENA/kg 20 50 70 100

Total

Time of death Hepatocellular carcinomas Number of animals among survivors Acute death Survivors (months)

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20

19

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694

E. Scherer and P. Emmelot

above 70 mg DENA[kg. Most interestingly, in a number of former cases, a clear topological association between small tumours and (remnants of) island tissue was observed. Figures 7 and 8 are typical illustrations of the presence of a cluster of glycogen-free tumour cells closely associated over a large part of its periphery with an island cell population deficient in ATPase but containing glycogen. These associations were found in the liver of rats which were moribund from mammary tumour--the common cause of death of Sprague Dawley females--and in which liver tumours had not yet reached full or lethal size.

DISCUSSION Mechanism of island cellformation Foci of liver cells (islands) characteristically and permanently changed in phenotypic markers and growth behaviour are induced by a single application of a hepatocarcinogen such as DENA [5]. Island formation may represent a sensitive system for studying and assaying a stable precancerous alteration and the process of liver carcinogenesis. Although in general "precancerous" is a rather loose term, it is applied here to a well-defined cell type, the island cell, which according to previous results may serve as a precursor in the process of liver turnout formation [7]. In the present experiments direct proportionality between number of islands induced and DENA in the dose range of 0'3-30 mg[kg for single application and regenerating liver, and 0"9-3"6 mg/kg day for multiple application and intact liver, was obtained. In these dose ranges no toxic effects on the liver were observed. The formation of N-7-ethylguanine in the DNA of the regenerating liver system also showed direct proportionality to the doses studied, i.e. 110 mg DENA/kg [16] ; a similar result has been reported for dimethylnitrosamine [17]. N-7-ethylguanine, and[or other ethylated products of DNA which may be expected to exhibit a similar dose dependency [18]--, might be instrumental in the action of DENA at the genetic level in carcinogenesis [19, 20]. From these kinetics the conclusion is drawn tlaat the conversion or "transformation" of a liver cell into an island-forming cell, which then grows out into a microscopical island, most probably occurs by a one-hit process, i.e. by one specific alteration produced by DENA at a critical site of its target cell. The persistently changed phenotype, the very low probability of the induction event (after partial hepatectomy one liver cell per 106 being transformed per

mgDENA/kg) and the underlying one-hit mechanism suggest that the island cells are permanently altered at the genetic level. Accordingly, the island cell type could result from one specific alteration produced by DENA in the DNA of a liver cell. Partial hepatectomy profoundly increases the number of islands produced by DENA [5]. According to the present view, DNA synthesis triggered by the operation would serve to fix the one-hit structural alteration in DNA, responsible for island cell formation, as a heriditary property prior to its repair [21, 22]. The one-hit kinetics pertain not only to the induction of ATPase-deficient islands but also to those islands which exhibit additional glucose-6-phosphatase deficiency and/or glycogen retention after fasting. This would indicate that the hit may be scored in a DNA segment that, either directly or indirectly, informs or controls various cellular properties. The relative and absolute decrease in the number of islands induced by a single application of > 50 mg DENA/kg may have various reasons. The diminuation of the response might result from toxic, viz. lethal or sterilising, hits accumulating in cells which have received the "transformation" hit. Secondly, the metabolic capacity of the two-thirds hepatectomized liver to convert DENA into its reactive and biologically active product (C2H5 +) may be a limiting factor in the case of these higher DENA concentrations. Under the latter conditions, the activity of the microsomal N-deethylating enzymes (which convert DENA to reactive metabolite) may even be further restrained by "self-poisoning" so that a disproportion of the DENA is excreted or metabolized elsewhere in the body [23]. Thirdly, toxic signs in the regenerating liver begin to appear at 50 mg DENA/kg (Fig. 1) and since regeneration enhances island formation [5], impairment of regeneration (DNA synthesis) will decrease island formation. The island-forming cell and its progeny, the island cells, have apparently acquired the capacity to proliferate, at least in partial independence of the growth control operating in liver [5]. The "growth autonomy" of the island cells is not absolute since island cells induced in intact liver react to partial hepatectomy with increased proliferation [24]. Thus, the regenerative Iesponse stemming from the toxic effect of DENA may also speed up island cell growth, as in the case of the 14 mg[ kg day dose administered to intact rats (Fig. 5). The situation in the regenerating liver of rats receiving a single toxic application of 70 mg

Fig. 7. Close tumour-island association detected in the liver of a non-fasted rat by A TPase staining (a, b). The tumour (trabecular pattern, glycogen deficient (c), basophilic staining (cresylviolet ) evenly distributed over the cytoplasm (d)) is in close contact with island cells (less deficient in A TPase staining than the tumour cells (a, b), PAS positive (c), normal basophilic staining of the cytoplasm (d) ) over a large part (a, arrows _~) of its periphery. Two other islands in (a) are indicated by arrows ,~.~ (a, x 30; b-d, x 120).

(to face p. 694)

Fig. 8. Close tumour-island association detected in the liver o]" a fasted rat by PAS staining (a, b). The PAS negative tumour shows close contact with island cells (PAS positive (b), A TPase deficient (c), less basophilic than normal liver cells (d) ) over a large part of its periphery (a, arrows). The surrounding liver is compressed by the tumour (as x 3 0 ; b, x 120).

Kinetics of Induction and Growth of Precancerous Liver-Celt Foci, and Liver Tumour Formation

DENA/kg or higher may be basically similar but more complicated. Regeneration in these livers is impaired but since the liver weight is fully restored in the course of time, the process should be more protracted as compared with livers subjected to lower (relatively) non-toxic doses of DENA. This more protracted regenerative response may afford an extra growth stimulus to the island cells leading to the observed increase in their numbers (Fig. 4). These effects are likely to [~e involved in the carcinogenic process since the rate of liver turnout formation has previously been shown to be a function of the total number of island cells present in the liver [7]. Accordingly, toxic effects of carcinogens below the cytolethal level may play an important role in carcinogenesis by providing a growth stimulus for precancerous cells. Mechanism of liver tumour formation Liver tumours are induced by a single application of _---50 mg DENA/kg to partially hepatectomized rats [21]. In these livers and in livers of rats receiving one subcarcinogenic dose island cells of similar morphological and enzyme histochemical phenotypes arise and proliferate, although at different rates. The island cells induced by a subcarcinogenic dose result from a one-hit process and, as demonstrated previously [7], such cells may serve as substrate for further carcinogenic action-apparently by receiving additional, i.e. sequential hits--to yield liver tumours. Combining these observations may lead one to suppose that one way in which tumour ceils could be formed by a single application of _-> 50 mg DENA/kg is from liver ceils which have received at least 2 concomitant hits, one conferring island-cell character and one allowing such cells to progress in time to malignancy without the need for further carcinogen application. Some morphological support for this view is offered by Fig. 7 and 8, in which tumour cells seem to arise focally in close association with island tissue, although the islands per se most probably are of clonal origin [6]. The focal progression in the absence of carcinogen, i.e. the quasi-

spontaneous development of heterogeneity in a seemingly homogeneous island cell population [25], could be caused by a small decrease in the fidelity of DNA replication [26, 27]. This decrease may result from a mutation affecting the DNA polymerase [26, 27], induced by a second hit as discussed above. However, decreased fidelity of DNA replication might also be part of the complex of changes (glucose6-phosphatase deficiency, glycogen retention, ATPase deficiency, etc. [5]) observed in islands induced by only one specific hit. In this case, as compared with the situation of a subcarcinogenic dose, only the greater number of proliferating island cells would decide the malignant outcome, since it would favour the establishment of the critical error required for progression. A third possibility to explain progression is that the normal background ("noise") infidelity of DNA replication might suffice for progression of island cells, again provided that enough proliferating cell at risk and time would be available. For its operation each of these mechanisms requires DNA synthesis not only for the production of a large number of cells at risk but also for making an error in a critical gene segment and perpetuating that error by another round of DNA synthesis in the'face of DNA repair. In addition, the randomness of the genetic changes resulting from infidel DNA replication may explain the general finding [4] that divergent tumour types do arise in liver and that similar types may be antigenically different [28]. Finally, it may be concluded that the wellknown enhancing effect of cell proliferation (DNA synthesis [11, 12]) on carcinogenesis is exerted at and beyond the induction stage proper, increasing the number of both the precancerous cells induced and their progeny which constitutes the population at risk to progress to malignancy, and favouring the latter transition.

Acknowledgement--We

wish to thank Miss B. Miilder and Mrs. M. van der Steen'Kamerbeek for their competent and careful technical assistance.

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1. 2. 3.

695

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