Rapid development of hepatocellular neoplasms in aging male C3HHeNCr mice given phenobarbital

CancerLetters. 39 (1988) 9- 18 Elsevier Scientific Publishers Ireland Ltd.

9

RAPID DEVELOPMENT OF HEPATOCELLULAR NEOPLASMS AGING MALE C3HIHeNCr MICE GIVEN PHENOBARBITAL*

JERROLD

M. WARD**+, PETER LYNCHb, and CHARLES

IN

RIGGSc

“Tumor Pathology and Pathogenesis Section, Laboratory of Comparative Carcinogenesti, Division of Cancer Etiology, National Cancer Institute, and bProgram Resources, Inc., and cInformation Management Services, Inc., NCI-Frederick Cancer Research Facility, Frederick, MD, 21701-1015 Kl.S.A.I (Received 14 September 1987) (Accepted 1 October 1987)

SUMMARY

One hundred and nineteen male C3H/HeNCr mice, 12 months of age, with spontaneous preneoplastic and neoplastic hepatocellular lesions were given phenobarbital (PB) at 500 ppm in drinking water. Groups of 9 - 10 mice were sacrificed after 12, 24 and 36 weeks of PB exposure. Identical numbers of untreated controls were used. A group of 6-week-old C3H/HeNCr mice were also given PB and sacrificed at 12.24 or 36 weeks. In aging mice, PB exposure significantly increased the number of gross tumors or microscopic foci, adenomas or carcinomas per liver at all time periods, especially unique eosinophilic proliferative lesions, while young mice did not develop any focal proliferative lesions by 36 weeks. These findings suggest that in aging mice a fraction of the hepatocyte population (normal, spontaneously-initiated or preneoplasticl is more highly susceptible to phenobarbital ‘carcinogenesis’ than are hepatocytes of younger mice. Key words: Mouse Phenobarbital

liver

tumors

-

Tumor

promotion

-

C3H

mice

-

INTRODUCTION

The mouse liver has been shown to be a frequent and important target site of carcinogenesis by a wide variety of chemicals [16,29,43]. Although these agents include many carcinogens that have the ability to cause mutations, transform *Research sponsored, at least in part, by the National Cancer Institute, DHHS, under contract NOlCO-23910 with Program Resources, Inc., and NOl-CO-23912 to Information Management Services, Inc. **To whom reprints should be addressed. 0 1988 Elsevier 0304-38351881t03.50 Published and Printed in Ireland

Scientific Publishers Ireland Ltd.

10

cultured cells and damage DNA [16], the mouse liver seems to be uniquely sensitive to other carcinogens that have not been shown to be genotoxic [43]. It is hypothesized that these so-called ‘non-genotoxic carcinogens’ [47] are ‘weak classical tumor promoters, and may appear carcinogenic for carcinogens, rodents used in safety assessment experiments by enhancing spontaneous hepatocarcinogenesis [33,39,44], especially mouse liver cells susceptible to Haru.s oncogene activation [30]. Phenobarbital (PB) has been established as a tumor promoter for mouse and rat liver when given after tumor initiation by carcinogens [6,24,26,42,45]. PB, itself, also induces rodent liver tumors in laboratory studies after l-2 years [9,13,25,28]. The development of these tumors usually occurs late in the experiment towards the end of the life span of the rats or mice. It has been suggested that PB and other carcinogens which have not been found to have mutagenic, cell transforming or DNA-damaging activity in vitro, may act by promoting the growth and progression of spontaneous preneoplastic lesions or tumors [33,39,41,44]. We have developed in vivo rodent models to study the effect of PB on naturally-occurring preneoplastic and neoplastic hepatocellular lesions in rats [39,44] and in this paper, in mice. MATERIALS AND METHODS

One hundred and seven male C3H/HeNCr (MTV - 1 mice were obtained at 4 weeks of age from the NC1 Mammalian Genetics and Animal Production Branch, Division of Cancer Treatment, Frederick, MD, and maintained 5/cage until 52 weeks of age on Purina 4% stabilized laboratory meal and water ad lib. At 52 weeks of age, 10 mice were sacrificed to evaluate the number and nature of hepatocellular lesions. Subsequently, 60 mice were placed on drinking water containing 500 ppm of PB (Sigma Chemical Co., St. Louis, MO) and 60 received no PB. At 12.24 and 36 weeks after initiation of exposure to PB, groups of 910 mice were sacrificed and necropsied. Thirty 6-week-old male C3H/HeNCr (MTV’- 1were given PB for up to 36 weeks, and groups of 10 were sacrificed at 12.24 or 36 weeks and their livers treated as above. Livers were weighed and the number of grossly visible hepatic lesions recorded. Two representative sections were evaluated histologically from each liver lobe. Focal hepatocellular proliferative lesions (FHPL), including microscopic hyperplastic foci, adenomas and carcinomas [14,38,40,42,45,46] were evaluated with a quantitative image analysis system (Videoplan, Zeiss, Inc., New York, NY) using Zeiss stereology programs [42,45]. The number of FHPL per liver was determined by multiplying the number of FHPL per cm3 by the liver weight of each mouse. Statistical analyses were performed using the non-parametric Jonckheere test [18] for positive trends over time and Wilcoxon rank-sum test [18] for significances of differences between groups. Because of the variable nature of much of the raw data being analyzed, non-parametric ranking methods were chosen over their parametric counterparts (i.e. regression and t-tests [35], respectively) to avoid the usual assumptions of normality and equal variances.

11 RESULTS

Liver weights, number of gross liver lesions and of histologically confirmed FHPL per unit area, volume of liver and mean volume of all types of FHPL were time- and treatment-related (Tables 1 and 2). At the beginning of the experiment (week 01, 4/9 control mice had microscopic foci and 4/9 had adenomas. PB increased significantly the liver weights (Table 1). numbers of total FHPL (Table 2) and of eosinophilic adenomas (Table 3) per liver, especially at 24 and 36 weeks. The increase in liver weights appeared to be due to significantly increased numbers of FHPL and increased size of lesions (Fig. 1). in all PB-exposed mice. Centrilobular cytomegaly was also present Histologically, FHPL were generally composed of hepatocytes with eosinophilic or basophilic cytoplasm, which has been demonstrated to be a reflection of the quantity of smooth or rough endoplasmic reticulum or ribosomes present [42]. The former usually contained large eosinophilic cells (Fig. 2). Much of the increase in number of FHPL per liver in PB-treated mice was due to the eosinophilic FHPL, the proliferative lesion usually induced by PB in mice and rats [6,13,39,42,44]. PB did not affect the number of basophilic FHPL. Neither mortality, carcinomas as percent of total liver tumors (adenomas and carcinomas) nor metastatic rate (Table 3) was increased by PB. Twenty PB-treated mice that died during the experimental period survived a median of 19 weeks, while 19 controls similarly survived a median of 17 weeks. FHPL were not found in any C3H mice treated with PB from 6 weeks of age for up to 36 weeks, although centrilobular hepatocytomegaly was seen. TABLE

1

GROSS HEPATIC PHENOBARBITAL Parameter and treatment’

FINDINGS

Experimental 0

Liver/body weight ratio (c/cl PB Control 6.5 + 0.3 Liver weight(g) PB _ Control 3.0 f 0.2 Gross liver tumors (no.1 PB Control 1.0 f 0.2

IN

AGING

MALE

C3H/HeNCr

MICE

EXPOSED

TO

weeks 12

24

36

0-36b

8.2 + 0.2’ 8.3 f 0.5

11.9 f 0.9d 5.6 2 0.4

19.2 -c 2.16,’ 8.7 2 1.7

15.3 z? 1.8 9.0 f 1.5

3.8 f 0.1 3.7 + 0.2

4.9 2 0.3’ 2.3 f 0.2

7.3 2 0.8* 3.3 + 0.5

3.6 f 0.5 2.5 f 0.6

7.5 + 0.8d 3.4 & 1.0

6.8 f 0.5d.e 3.0 f 0.6

* There were groups of 9- 10 mice at sacrifice times of 0,12.24 and 36 weeks. b Mice dying or sacrificed in moribund condition during the experiment. c Mean f S.E.M. d P < 0.01 vs. control Wailed). e P < 0.05 trend test, by time (l-tailed). r P < 0.05 vs. control (e-tailed). g Not determined, usually coalescing masses.

5.9 -t 0.54 3.6 -t 0.4 ND% ND

PB Control PB Control

-

4.0

1.0

0.1

1.2 f -

0.6

0.6 f 0.2

5.2 f 2.4 -

58.1 f 28.0 -

7.2 f -

2.1 f -

0.4 + -

13 -

128.1 f 52.1

0

Experimental week

3.9 3.9 4.0 98.1

83.2 11.0 3.0 23.0 82.8 f 1.6 2 1.3 f 1.8 + 44.3

-t 19.7 + 2.9 -c 1.3 f 8.5 f 69.9

66 48 1.6 f 0.2b,C 1.0 + 0.2 3.7 f 0.6 2.4 f 0.6 14.2 f 2.5 9.3 f 2.5 47.7 f 14.0

12

9.7 4.2 234.4 70.2

124.3 17.2 2.5 84.5 144.9

47.5 3.0c 1.0 20.3 54.7 f 3.5 f 1.8 2 133.7 f 64.4

f + f f *

125 34 2.8 2 0.3’ 1.1 f 0.3’ 5.2 2 0.9’ 2.8 rt 1.0 21.1 f 3.7c 6.6 * 2.0 114.6 f 21.0

24

62.4 3.8,d 2.0 63.0”d 34.7

0.7 1.3 3.5C,d 2.8 51.3d

0.3e,d 0.2”

f 1.5 f 4.7d & 165.6 f 101.1

k f * f f

136.4 26.1 7.0 183.0 52.0 5.8 11.6 350.4 212.6

f f f f f

171 54 2.6 f 1.6 f 4.5 4.5 29.6 12.9 186.4

36

’ Groups 9-10 mice were sacrificed at each time period. Week 0 is 52 weeks of age. FHPL includes all microscopic hyperplastic foci, hepatoceliular adenomas and carcinomas, including those characterized morphologically as eosinophibc, basophihc, mixed clear and vacuolated. b Mean f S.E.M. c P < 0.05 vs. control (&tailed). d P < 0.01 trend test, by time (l-tailed).

Focus (ah types) Volume (mm%) Eosinophilic FHPL/Iiver Eosinophiiic focus volume (mm*) Basophiiic FHPL/Iiver Basophilic focus volume

FHPL/Iiver

FHPLlcm’

Control PB Control PB Control

PB Control PB Control PB Control PB Control PB

No. of FHPL

FHPL/cme

Treatment’

Parameter

FHPL IN AGING MALE C3HIHeNCr MICE

TABLE 2

619 (67) 919 (100) 9/10 (90) lO/lO(100) 8/10 (80) lO/lO(100) 8/10 (80) 17120 (85) 15/19 (79)

0.8 f 0.8 6.8 f 1.8’6 2.9 f 1.3 15.1 + 2.1d.e 2.2 f 0.9 21.6 f 3.6d,” 3.8 f 1.3 ND ND l/9 (11) o/9 (0) 3110(30) 3/10 (30) l/10 (10) 5110(50) 5/10 (50) lo/20 (50) 11/19(58)

Carcinoma

l

Includes microscopic hyperplastic foci, hepatocellular adenomas and carcinomas. No. effect/no. at risk (0~). b Mice dying or sacrificed in moribund condition during the experiment. c Mean * S.E.M.; ND, not determiend. d P < 0.01 trend test, by time (l-tailed). * P < 0.01 vs. control (&tailed).

619 (67) 9i9 (100) 9/10 (96) lO/lO (100) 8/10 (80) lO/lO (100) 10110(100) 19120 (95) 18/19 (95)

Control PB Control PB Control PB Control PB Control

0 12 12 24 2 36 36 0-36b o-36b

Eosinophilic adenomas/Iiver

HepatoceIIulartumors Adenoma

FHPL’

Treatment

Experimental week

l/6 (16) O/36 (0) 4136(11) 4i95 (4) 2127 (7) 81142(6) 5135 (14) ND ND

Carcinomas/ total tumors (0~)

INCIDENCEOF FOCAL HEPATOCELLULAR PROLIFERATIVE LESIONS IN AGING MALE C3HIHeNCrMICE

TABLE 3

019 (0) 019 (0) 0110 (0) O/l0 (0) l/10 (10) o/10 (0) o/10 (0) 3120(15) l/l9 (5)

Pulmonary metastasis

Fig. 1. Gross tumors in liver of Wweek-old untreated male C3H/HeNCr mouse (on left) and 88-weekold mouse exposed to PB in the water for 36 weeks (on right). Note many coalescing tumors in liver of PB-treated mouse and fewer tumors in control.

Fig. 2. Portion of eosinophilie hepatocellular eosinophilic cytoplasm. H & E, X 250.

adenoma

showing

large

tumor

cells

with

pale

15 DISCUSSION

Our findings clearly show that preneoplastic and neoplastic hepatocellular lesions develop quickly in aging C3H mice during exposure to PB. FHPL appear in increased numbers as early as 12 weeks, in marked contrast to fewer and smaller lesions that develop during similar periods of PB exposure that began in young mice of various strains including C3H [6,9,10,25,82]. Our study suggests that PB, as its major effect, either induced new preneoplastic and neoplastic eosinophilic lesions de novo or hastened the progression from latent initiated or susceptible hepatocytes. This hypothesis is supported by several pieces of evidence: (11 many more eosinophilic FHPL appeared in our C3H/HeNCr mice given PB than in untreated controls; (2) basophilic FHPL were not affected; and (31 natural tumor progression to carcinoma, metastases, and mortality were not obviously affected by PB in our study or others [13]. Aging of rodents is generally associated with a change in response to chemical carcinogens and xenobiotics [1,2,12]. Aged rodent tissues may be more or less susceptible to chemical carcinogenesis. This aging change is due, in part, to alterations in metabolism [15,37]. In the case of PB, we have shown that aging F344 rats were more susceptible to the induction of preneoplastic and neoplastic hepatocellular lesions than were young rats [39,44]. Mice given a phorbol ester were less sensitive to its promoting effect as they aged, however [36]. Evidence exists that PB is metabolized less efficiently in aging rats [20,31] while conflicting results have been reported in mice [19,22]. We saw no increased toxicity of PB in our aging mice to suggest that PB pharmacokinetics may have been responsible for higher levels in blood. Aging mice do, however, show signs of increasing brain sensitivity to PB with age [22]. A similar effect is suggested for response of hepatocytes to its carcinogenic effect. PB has been shown to exert a proliferative effect on normal rat hepatocytes in vitro [3,23] and focal proliferative hepatic lesions in rats initiated with nitrosamines [4,32,34]. A direct effect of PB also was shown to stimulate gamma-glutamyl transferase activity in cells of spontaneous mouse liver tumors [49]. On the contrary, our findings support the hypothesis that the major effect of PB was to increase the numbers of initiated cells that progressed to preneoplastic and neoplastic eosinophilic FHPL or promoted the progression or clonal expansion of previously spontaneously initiated or PB-susceptible cells to eosinophilic FHPL, perhaps through activation of the Ha-ros gene or promotion of hepatocytes with aged-related activated Ha-ros gene [30,50]. This hypothesis is supported by the fact that both control and PB-treated mice died at the same rate, with no dramatic effect on the normal progression of natural liver tumors, especially those of the basophilic type, and that the major effect of PB was the ‘induction’ of eosinophilic FHPL. The possible lack of effect by BP on natural tumor progression is comparable to that of phorbol esters in skin tumor promotion studies in mice [1’7], but in marked contrast to its effects on progression of preneoplastic and neoplastic hepatic lesions in rats or mice after initiation [11,21,26,27,45,48] or mice after initiation and promotion by other

16

agents [10,45]. It was recently demonstrated that liver tumor progression in mice may be affected by a single gene in a strain with a low spontaneous incidence of liver tumors [8]. This new system may be a model for studying the role of tumor promoters in tumor progression. This is especially valid using that authors’ liver tumor classification scheme [5,7]. ACKNOWLEDGMENTS

We would like to thank Dan Logsdon, Teddy Thompson, Kathy Breeze, Joyce Vincent, Barbara Kasprzak, Rick Klabansky, and Dee Green for excellent technical assistance and Drs. Bhal Diwan, Aki Hagawara, Lucy Anderson and Jerry Rice for valuable critique. REFERENCES

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