liver regeneration and induction of hepatomas in mice by ...32 hr followed soon afterwards by the...

[CANCER RESEARCH 30, 2685-2690, November 1970]

Liver Regeneration and Induction of Hepatomas inMice by Urethan1

I. N. Chernozemskr and G. P. Warwick3

Chester Beatty Research Institute, Institute of Cancer Research, Royal Cancer Hospital, Fulham Road, London S. W, 3, England

SUMMARY

The pattern of regeneration and cell proliferation wasstudied in liver remnants of B6AFi hybrid mice during thefirst 85 hr following partial hepatectomy. On the basis ofthe estimated timing of DNA synthesis and mitoses, maleand female adult mice were inoculated once with urethan, 1mg/g body weight, at the following times after partialhepatectomy: 4 to 5 hr, i.e., at an early prereplicativeperiod; 17 to 18 hr, during a late prereplicative period; 31to 33 hr, shortly before the onset of extensive DNAduplication; and 46 hr, before the peak of mitoses.

The hepatoma incidence showed a clear-cut relationshipbetween the timing of urethan treatment in relation to priorhepatectomy. After 13 months, 10, 68, 42, and 77% of themale animals bore at least one hepatoma, compared with3.3% among untreated males, 6% in those treated only withurethan, and 19.4% following hepatectomy alone. Hepatictumors were rare in the females. In urethan-treated mice,body weight was reduced by up to 50% of that of controls.

INTRODUCTION

This experiment was designed further to elucidate possibledifferences in the ability of a chemical to produce hepa-tomas when administered to adult animals with intact orregenerating livers. That such differences may exist isprovided by the fact that some chemicals such as urethanalthough capable of producing cancer at various sites do notin general induce hepatomas in adult mice and rats, but doso when given as a single application at birth or soonafterwards (5, 7, 25). Also, it has been demonstrated recently that prior partial hepatectomy can substantially increasehepatoma yield in several strains of adult rats (17, 30, 31)and mice (13, 16, 21) when different chemicals are appliedfollowing the operation. However, in these experiments thedose, the timing of the applications, and the tumor yields

'This study was supported by a grant from the Medical ResearchCouncil and British Empire Cancer Campaign for Research.

2This work was carried out during a Research Fellowship of theInternational Agency for Research on Cancer, Lyon, France, while onleave from Oncological Research Institute, Sofia 56, Bulgaria.

3To whom reprint requests should be addressed at Chester BeattyResearch Institute.

Received March 27, 1970; accepted July 14, 1970.

varied considerably. Thus there is no information as topossible variations in the susceptibility of the regeneratingliver to the carcinogenic action of any chemical given atdifferent times during the first wave of proliferation following the operation. We have thus studied the possiblehepatocarcinogenicity of a single dose of urethan administered to adult B6AFj mice following two-thirds partialhepatectomy at predetermined times corresponding to theperiods in parenchymal cells of early prereplication, lateprereplication, DNA replication, and mitosis. Urethan waschosen because of its weak hepatocarcinogenic activity inadult BÃ’A?! mice and its high hepatocarcinogenic activity inneonatal mice of this strain resulting from only a singleinjection, its short life of 7 to 8 hr in adult mouse liver (6,20), and its minimal damaging effects on liver parenchyma(4).

MATERIALS AND METHODS

Mice. A group of 526 male and female young adult B6AF,mice were used. Their average weight was 26.5 Â±2.9 g formales and 20.4 Â±2.6 g for females. Parental A males werekindly supplied by the Laboratory Animal Center, MRCLaboratories, Carshalton, Surrey, England, and C57BLfemales were from this Institute. Mice were kept understandard conditions and fed, ad libitum, Diet 86, fromPlowco Feeds Ltd., London, England.

Partial Hepatectomy. The method of Higgins and Anderson(12) for rats was adapted for mice. It was estimated that ourprocedure led to the resection of 66.5 Â±3.1% of the totalliver weight; the weight of the remnants was 29.2 Â±2.1%,while that of the pieces above the ligature was 4 to 5%. Theoperation was performed under light ether anesthesia andsterile conditions. Hepatectomies were timed to avoid diurnalfluctuations. For 24 hr after the operation, the animals weregiven 5% glucose for drinking. Only mice going throughoperation without complications, showing quick recovery,with a maximum drop in body weight of 8%, and withoutjaundice were further used.

Pattern of Regeneration. Groups of 5 to 7 mice werepartially hepatectomized at 6-hr or, in a few cases, 12-hrintervals. One hr prior to killing they were given injectionsi.p. of 0.75 i/Ci/g of thymidine-3H (specific radioactivity, 5

Ci/mmole, Radiochemical Centre, Amersham, England). Partsof each liver were quickly frozen in liquid nitrogen and usedfor isolation of DNA. Right lobes were fixed in 10% neutralformalin or alcohol:formalin:acetic acid mixture. Paraffin

NOVEMBER 1970 2685

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/. N. Chemozemski and G. P. Warwick

sections were stained with hematoxylin and eosin for generalsurvey, or with periodic acid-Schiff reaction, methyl green-pyronin (controls treated with RNase), Masson's van Gieson

variant (19), and for reticulin; formalin-fixed sections werestained for neutral fat with Oil Red 0. For radioautography,paraffin sections were covered with Kodak AR 10 films,exposed at 4Â°for 15 to 18 days, developed in Kodak D19

developer, fixed, and stained with hematoxylin and eosin orFeulgen stain. Samples were also fixed in Karnovsky's fixa

tive and examined under a Siemens electron microscope.Parallel scoring of labeled and mitotic nuclei was done in anocular square frame at X760. The specific radioactivity ofisolated DNA was measured as described before (22) in aPackard 3375 liquid scintillation spectrometer, efficiencybeing calculated by the channel ratio method.

Urethan Treatment and Tumor Survey. Urethan (ethylcarbamate, May and Baker, Ltd., Dagenham, England) wasused as a fresh 6% aqueous solution. Groups of partiallyhepatectomized mice each received a single injection at 1 of4 times (Table 1). Control groups received either 1 or 3mg/g body weight. Those mice that recovered well wereregularly weighed and inspected. Most of the experimentalmice were killed 1 year after treatment; some controls andthose mice submitted only to hepatectomy were killed later(Table 1). These were weighed and autopsied, large tumormasses being considered as a single lesion, and measured.Specimens were also taken from apparently normal livers,from some lungs with tumors, and from all other lesions.

They were fixed, sectioned, and stained as for regeneratinglivers.

All quantitative data were evaluated statistically and thesignificance between mean values was determined with the ttest.

RESULTS

Liver Regeneration. During approximately the first 10 to15 hr after hepatectomy, there was a progressive vacuoliza-tion, fat infiltration, gradual depletion of glycogen, dissolution of basophilic bodies, and depleted diffuse pyronino-philic material in the cytoplasm of parenchyma! cells. Earlyvascular lesions, hemorrhages, and necrotic foci were alsoscattered in the parenchyma. Very few cells were labeled ordivided. This stage corresponds to the early prereplicativeperiod suggested recently by Baserga (1).

At 17 to 18 hr, the late prereplicative period, the majorityof these changes persisted except that reappearance ofRNase-sensitive material in the cytoplasm corresponded to anobvious increase of ribosomes attached to membranes. Theuptake of thymidine-3H was still lower than in control livers

and very few cells were labeled.There was a substantial increase of labeled cells and a rise

in the specific radioactivity of isolated DNA at about 30 to32 hr followed soon afterwards by the peak of DNAsynthesis at 40 hr for males and at 46 hr for females, whenabout one-third of the parenchyma! cells were labeled (Chart

Table 1

Hepatoma yield in male and female B6AF] miceThe incidence of hepatomas in untreated controls (Group 1) was compared with those partially

hepatectomized (Group 2), given i.p. injections of urethan (Group 3), or given urethan at 4 differenttimes after hepatectomy (Groups 4 to 7). Very large tumor masses were considered as a single lesion.

Group ModeofNo.1234567treatmentUntreated

controlsPartial

hepatectomyUrethan6Partial

hepatectomy+urethaninjectedafter

4 to 5hrPartialhepatectomy+

urethaninjectedafter17 to 18hrPartial

hepatectomy+urethaninjectedafter

31 to 33hrPartialhepatectomy+

urethaninjectedafter46 hrSexMFMFMFMFMFMFMFÂ»i

eNo.otanimaisexamined3030312235202017251526162615Mean

agea(wk)Sincebirth6678737366656666696964706468Sincetreatment656557565656565656565353No.10602021171112200Mice

withhepatomas%3.319.46.010.05.968.06.642.012.577.0Average

no./mouse1.01.31.01.01.01.91.01.21.01.5Averagediameter(mm)5.010.05.05.04.09.23.07.55.010.0

"Mean age at the end of the experiment.0Hepatoma yield was similar in the groups given injections of 1 and 3 mg/g body weight of urethan.

2686 CANCER RESEARCH VOL. 30



Liver Regeneration and Urethan Corcinogenesis

1). The isolated DNA revealed the same time pattern oflabeling as did the scoring (Chart 2).

The high level of mitosis began at 8 to 10 hr after theonset of DNA synthesis and the mitotic rate curves weresimilar in shape to those based on the labeling index (Chart3). The rate of DNA replication and of mitoses thendeclined gradually, and from then on there was a mixedpopulation of parenchymal cells in the late prereplicativestage, in DNA replication, and in mitosis. All nonparen-chymal cells commenced their cycle of replication after theparenchymal cells (Chart 1, "other" cells).

100-

350-

- 300-

-fa 250

8.I 200

| 150

II

SO

Males Ftmalts

ParenchymalcellsOthercelliMean

values2râ€¢â€¢--â€¢*---Â»**0â€”0Ã›â€”Ã›t

*

Controls 26 3l 10 16 51 57 63

Hours after hepatectomy

Chart 1. Thymidine- H-labeled nuclei of the parenchymal and"other" liver cells during the first 85 hr following partial hepat

ectomy in male and female B6AP! mice. Kodak AR 10 strippingfilm method. Data points, group means; vertical bars, S.D.

20

S1"

Males

Females

Controls 26 31 10 16 51 57


Chart 2. Specific activity of DNA isolated from pooled male andand female B6AFi mouse livers taken during the first 85 hr followingpartial hepatectomy. Thymidine-3H, 0.75 juCi/g body weight, was

injected i.p. 1 hr before the animals were killed. DPM numbers are tobe multiplied by 10 to obtain accurate values.

Controls 26 31 10 16 51 57 63


Chart 3. Parenchymal liver cell mitoses during the first 85 hr afterpartial hepatectomy of the same mice as in Charts 1 and 2. Nuclei inlate prophase, metaphase, anaphase, and early telophase were scored.Data points, group means; vertical bars, S.D.

Mortality of Animals and Body Weights. All of the untreated mice survived except one 9-month-old female thatdied bearing leukosis. In the hepatectomized groups, 2 malesdied 10 months postoperatively, 1 with pneumonia and 1with a large abdominal sarcoma. Another died after 12months, bearing a massive hepatoma. After hepatectomyfollowed by urethan, the highest death rate (14%) wasamong those given injections at 4 to 5 hr. The mortality was4, 8, and 0% in the groups given injections at 17 to 18, 31to 33, and 46 hr. Subsequent mortality among these animalsvaried from 12 to 17% for males and from 4 to 15% forfemales. The majority of deaths were at 8 to 10 months;lung tumors, sarcomas, and leukosis were found in theautopsied mice. Some mice given injections of 3 mg/gurethan died after 8 to 10 months, although 82% surviveduntil the end of the experiment.

At the time of killing, 65 weeks, the body weights ofcontrols and of mice hepatectomized only were similar,being 50 Â±2.8 g for males and 42 Â±4.6 g for females. Thoseanimals receiving urethan alone increased in weight moreslowly, the weight difference becoming more apparent at theend of the experiment, at 37 g for males and 36 g forfemales. In the animals receiving urethan after partial hepatectomy, increase in body weight was also retarded, theeffect being up to 50% in those mice given injections 4 hrafter the operation.

Tumor Yield and Survey. The incidence of hepatomas isshown in Table 1. Only lesions histologically examined andclassified as benign or malignant hepatocellular tumors areincluded. Hepatic tumors were single or multiple noduleswith diameter rarely less than 8 to 9 mm, centrally situatedin most cases. They were paler than the rest of theparenchyma with vessels on the surface. In all females,hepatomas were less numerous, smaller, and usually single.Microscopically, well differentiated and highly vascularizedtrabecular hepatomas were observed most frequently (Figs. 1and 2), but some of the tumors were composed of small,closely packed cells. In large tumors, widespread necrosis was

NOVEMBER 1970 2687



/. N. Chemozemski and G. P. Warwick

*

Fig. 1. Trabecular hepatoma on a male animal killed 56 weeks afteruiethan treatment given at 17 hr after partial hepatectomy. H & E,X 80.

EÂ¥

Fig. 2. Trabecular hepatoma approximating to normal liver withmany cells in mitosis. H & E, X 100.

seen. In the few histologically examined lungs and lymphnodes, no mÃ©tastaseswere found.

Besides hepatocellular tumors, a variety of other neoplasmswere found (14, 15, 23, 24, 26). Among them were foundregularly lung adenomas, lymphatic lymphoma and leukemia,Harderian gland adenomas, liver blood cysts, forestomachpapillomas, and 2 rhabdomyosarcomas.

DISCUSSION

While not hepatocarcinogenic for adult B6AFt mice following a single injection (Table 1, Group 3), urethan induced

multiple hepatomas in, up to 77% of adult male animalswithin 53 weeks when the injection was given at varioustimes following two-thirds partial hepatectomy (Groups 5 to7). It is unlikely that tumor production was due to adifference in the effective dose of urethan, since a 3-foldhigher dose did not increase tumor incidence in unoperatedadults. While the operation itself led to some increase intumor yield, the tumors appeared later in life than thoseafter urethan treatment and have not been observed at all inanother species (8). Thus, apparently urethan is a powerfulhepatocarcinogen when applied during the first wave ofproliferation of adult B6AFi mouse liver following partial




Liver Regeneration and Urethan Carcinogenesis

hepatectomy, the potency of the chemical varying dramatically according to the time at which the single dose wasadministered.

The regenerating mouse liver, in particular that of thisstrain, is very suitable for such an experiment, since therewas a considerable spread in the timing of the eventsobserved during liver replacement in contrast to thatobserved in other species (3, 9, 11). Also, there was asatisfactory synchrony of events in the liver of animals inthe same group, thus enabling the single injection to beadministered to groups in which livers were at a corresponding phase of regeneration. Very few parenchymal cellssynthesized DNA at a rate greater than controls before 27 to29 hr postoperatively in all the animals studied, enablinggroups to be given injections at the two phases of thisprereplicative period. Of these, mice given injections at 4 hr,i.e., during early prereplication, showed no increase in hepa-toma yield above the unoperated controls, while thosetreated at 17 to 18 hr, the late prereplicative phase, developed hepatomas in 68% of the animals. In fact, hepatomaswere produced in high yield in all groups of operated maleanimals when treatment took place at any time duringreplication, i.e., in the latter phase prior to DNA synthesis,during DNA synthesis, and during mitosis. The livers of thelast group contained a mixed population of cell typesincluding some in DNA synthesis and in the period prior tomitosis, in mitosis, and in preparation for a new wave ofproliferation.

While this work was in progress, Hollander and Bentvelzen(13) reported that whereas in C3H/H2A mice, in which thereis a high spontaneous hepatoma incidence, urethan producedhepatomas in 50% of the animals at 15 months, the yieldrose to 84% when urethan was administered 4 days after theoperation. This system might not be directly comparable toours, since the first wave of parenchymal cell replicationwould have virtually finished before the chemical was administered, while it would have been in contact with cells duringthe second wave of proliferation.

Since urethan may have to undergo metabolism to achemically reactive species such as a carbonium ion or a freeradical before combination with important cell constituentscan occur, one should interpret the present results, and thoseof others obtained with urethan, in the light of possibledifferences in the ability of the liver to perform such anenzymatic change at different times after partial hepatectomy. In this context, it has been well established thatimmediately following partial hepatectomy there is adecrease in the activity of almost all liver enzymes (2)corresponding to an impairment of many metabolic processes, and that the rate of restitution of activity variesbetween different enzymes (2). A further complication stemsfrom the report that, in the rat at least, the synthesis ofRNA starts at a higher rate almost immediately after theoperation (10) with a concomitant increase in RNA poly-merase activity (27, 28). If such a situation prevails in themouse, we cannot relate the absence of tumors in animalsgiven injections at 4 hr after hepatectomy with an absenceof RNA synthesis, although it was obviously much moreintense at 17 to 18 hr when a high tumor yield was

obtained. One must therefore balance the effects due topossible differences in the concentration of reactive metabo-

lite(s) formed from urethan with any subsequent interferencewith processes of replication such as macromolecular synthesis in the regenerating liver, which could lead to tumorformation.

The observed sex dependence of the hepatoma yield produced in female mice implies an important function of sexhormones in this system as has been found in others (5, 7,25).

Since castration can abolish this different sex response afterurethan treatment (18, 29), it appears that in this system thesex hormones exert their effects mainly on the process oftumor development and not at an earlier stage.

ACKNOWLEDGMENTS

We thank Dr. A. J. S. Davies, for valuable discussions during theearly part of this work and for providing help with facilities forpartial hepatectomies; Miss Elizabeth Beaumont, for the electionmicroscopical sections; and Mr. M. S. C. Birbeck for comments onultrastructure findings.

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1970;30:2685-2690. Cancer Res I. N. Chernozemski and G. P. Warwick by Urethan

Mice1Liver Regeneration and Induction of Hepatomas in B6AF

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