High sensitivity of the LEC rat liver to the carcinogenic effect of diethylnitrosamine

Cancer Letters, 51(1990)

247

247-250

Elsevier Scientific Publishers Ireland Ltd.

High sensitivity of the LEC rat liver to the carcinogenic effect of diethylnitrosamine H. Takahashi, Department

K. Enomoto,

ofPathology,

Y. Nakajima

and M. Mori

Sapporo Medical College, S.l,

W. 17,

Chuo-ku, Sapporo 060 (Japan)

(Received 25 December 1989) (Revision received 7 February 1990) (Accepted 6 March 1990)

Summary

Introduction

The initiation sensitiuity of the liver of the LEC (Long-Evans with a cinnamon-like coat color) rat, a new mutant strain with a high incidence of spontaneous liver tumors, was

The LEC rat is a new mutant inbred strain which shows a sudden onset of liver injurlr at around 16 weeks after birth [ 11. This hepatitis, characterized by submassive necrosis of hepatocytes with few inflammatory cell responses, occurs in all rats of this strain [2]. Although the pathogenesis of the hereditary hepatitis of the LEC rat has not yet been clarified, genetic analysis revealed that a single autosomal recessive gene was responsible for the appearance of the hepatitis [3]. The appearance of GST-P-positive foci was observed in the livers of LEC rats that recovered from severe hepatitis [4]. The number and size of these foci increased with age, and a high incidence of hepatocellular carcinomas developed in long-surviving LEC rats [5,6]. This natural history of liver tumor development in LEC rats is similar to that seen in human liver-cancer development. Thus, the LEC rat is a useful model for studying the mechanisms of human liver-cancer development. In this experiment, we focused on the initiation phase of LEC rat liver carcinogenesis. investigated whether the LEC rat liver is sensitive to a low dose of a

studied by treatment with diethylnitrosamine (DEN)

low

doses

of

coupled with modified Solt and Farber’s selection. LEC and control LEA (Long-Evans with an agouti coat color) rats received i.p. injections of 10 mg/kg of DEN, then selected by feeding with a diet containing 0.02 !% 2-acetylaminofluorene (AAF) for 7 days combined with partial hepatectomy (PI-f). The numbers of placental glutathione S-transjerase (GST-P)-positive foci

in the livers of LEC rats were 20 times higher than those in LEA rats. These results suggested a high sensitivity of the LEC rat liuer to the carcinogenic effect of DEN. The association

between

initiation

sensitioity

and

spontaneous liver-tumor development and the possible usefulness of the LEC rat for in vivo short-term discussed.

tests

of

hepatocarcinogens

Keywords: diethylnitrosamine; GST-P-positive foci. 0304-3835/90/503.50

LEC

are

rats;

0 1990 El sevier Scientific Publishers Ireland Ltd.

Published and Printed in Ireland

248

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I

2 onIs’

Group 3 AAF only

Group DEN

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B

1

D

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I

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B

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I

4

only

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*:*

1

B

J

B

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Fig. 1. Experimental protocols used in the present study are shown. In Group 1 (DEN - AAF + PH), 14 days after i.p. injection of 10 mg/kg of diethylnitrosamine (DEN), LEC and LEA rats were fed a diet containing 0.02% AAF for 7 days. On the 4th day of the AAF diet they were subjected to partial hepatectomy (PH). After completion of the AAF diet, they were placed on a basal diet (B) for 10 days and were killed at 31 days after initial treatment. Four control groups were prepared in LEC and LEA rat; i.e., Group 2 (AAF + PH alone), Group 3 (AAF alone), Group 4 (DEN alone) and Group 5 (PH alone).

hepatocarcinogen DEN, using modified Solt and Farber’s selection protocol. Materials and methods

Figure 1 summarizes the regimen for detecting initiated hepatocytes in the livers of LEC and LEA rats by a modification of the Solt and Farber’s method [7] as assayed by GST-Ppositive foci. Briefly, in Group 1 (DEN + AAF + PH), 14 days after i.p. administration of 10 mg/kg of DEN, rats were fed a diet containing 0.02% AAF (Japan Clea Co.) for 7 days. On the 4th day of the AAF diet, they were subjected to PH. After completion of the AAF diet, they were placed on a basal diet (Japan Clea Co.) for 10 days and killed at 31 days after the initial treatment. Initiated cells in the liver were assayed as GST-P-positive foci. As shown in Fig. 1, 4 control groups were prepared, i.e., Group 2 (AAF + PH alone), Group 3 (AAF alone), Group 4 (DEN alone) and Group 5 (PH alone).

Livers were fixed with ice-cold acetone for hematoxylin eosin staining and and immunohistochemical examination of GST-P. Staining of GST-P was performed by the avidin-biotin-peroxidase complex method as described previously [8]. The GST-P-positive foci larger than 0.01 mm2 were counted. The numbers per square centimeter and area (mm2) were measured by a Personal Image Analysis System LA-555 (PIAS, Japan). Results and discussion

As shown in Table 1, treatment of rats in Group 1 (DEN + AAF + PH) raised 10 times more GST-P-positive foci in the LEC rat livers than in LEA rats in both male and female. The average area of the focus in LEC rats was also larger than that in LEA rats. A small number of GST-P-positive foci was induced in the livers of both strains by a selection procedure with AAF and PH (Group 2, AAF + PH alone), in which the number of foci in LEC rats was significantly greater than that in LEA rats. In addition, only few foci were seen in the livers of LEC rats of Group 3 (AAF alone). In Group 4 (DEN alone) and Group 5 (PH alone), no GST-P-positive foci were seen in the livers of LEC and LEA rats. Solt and Farber’s selection protocol was used for an initiated cell assay in vivo [9], and GST-P was considered as the most reliable marker for the preneoplastic liver lesions [ 101. Thus, the results of the present study clearly indicate that the hepatocytes of LEC rats have quite high susceptibility to the initiation effect of DEN when compared with LEA rats, which develop no liver tumors in their natural history. Although the mechanisms of such high susceptibility to this carcinogen were not elucidated, the following possibilities could be postulated. First, since alterations of some drug-metabolizing enzymes in young LEC rats were reported [ 111, changes in the metabolism of DEN could result in more DNA damage. Alternatively, a reduced ability to repair DNA damage in the LEC rat liver can be postulated. With respect to the precise mechanisms of the

249

Table 1.

GST-P positive foci in the livers of LEC and LEA rats after selection of modified Solt-Farber protocol.

Treatment”

Strain

Sex

No. of animaW

Average size of foci (mm*)

No. of foci (/cm? 6.8’ 1.2 8.4 0.5

0.071 0.063 0.047 0.036

* f * *

O.OW 0.045 O.Ol& 0.030

ztzl.lc * 0.7 f 1.7’ -t 0.1

0.066 0.027 0.019 0.019

* f f zt

0.062 0.003 0.004 0.008

0.1 * 0.2 0.0 0.1 * 0.2 0.0

0.023 0.000 0.026 0.000

f 0.006

14 11 15 15

0 0 0 0

0 0 0 0

5 3 4 2

0 0 0 0

0 0 0 0

(1) DEN -, AAF + PH

LEC LEA LEC LEA

Male Male Female Female

10 11 6 13

15.2 1.4 19.2 0.4

(2) AAF + PH alone

LEC LEA LEC LEA

Male Male Female Female

13 8 11 5

1.8 0.6 0.7 0.2

(3) AAF alone

LEC LEA LEC LEA

Male Male Female Female

10 5 8 6

(4) DEN alone

LEC LEA LEC LEA

Male Male Female Female

(5) PH alone

LEC LEA LEC LEA

Male Male Female Female

f + -c &

zt 0.003

Values represent mean f SD. OEachtreatment is shown at Fig. 1. bAll of rats were killed at 12 weeks. cSignificantlydifferent from LEA rats, P < 0.01.

high initiation sensitivity of the LEC rat liver, further studies are necessary. In Group 2 (AAF + PH alone), a higher number of GST-P-positive foci was also observed in the LEC rat liver. This suggests that feeding with a 0.02% AAF diet for 7 days is enough to induce GSTP-positive foci in the LEC rat. This indicates the possibility of high sensitivity of LEC rats not only to DEN but also to AAF. Studies using other liver carcinogens are now in progress. In Group 4 (DEN alone), no GST-P-positive foci were apparent in the LEC rat livers by the

injection of a small dose of DEN. This indicates the importance of the following selection procedure for promoting the initiated cells to preneoplastic lesions even in carcinogen sensitive LEC rat. We have also examined the possibility of spontaneous proto-oncogene activation in the hepatocytes of the LEC rat, but there is no evidence of proto-oncogene activation as of now. Thus, it appears that LEC rat hepatocytes may possibly be initiated by weak liver carcinogens naturally available in the

250

The high incidence of environment. spontaneous liver tumors in LEC rats may be caused by the promotion of these initiated hepatocytes under the condition of hereditary chronic hepatitis (repeated cell death and regeneration). Since the majority of human cancers are considered to be caused by environmental carcinogens [ 121, it is of importance to develop system for such a sensitive detection environmental carcinogens. LEC rats may be used for short-term bioassays of weak environmental carcinogens. Acknowledgments The authors are grateful to Ms. K. Kagami and Ms. N. Kawano for their careful breeding of rats, and to Ms. M. Kamada and Ms. M. Kinoshita for technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research (63480145) from the Ministry of Education, Science and Culture, and Grants from the Princess Japan, Takamatsu Cancer Research Fund and the Uehara Memorial Foundation, Japan. References 1

2

Sasaki, M., Yoshida, M.C., Kagami, K., Takeichi, N., Kobayashi, H., Dernpo, K. and Mori, M. (1985) Spontaneous hepatitis in an inbred strain of Long-Evans rats. Rat News Lett., 14,4--6. Yoshida, M.G., Masuda, R., Sasaki, M., Takeichi, N., Kobayashi, H., Dempo, K. and Mori, M. (1987) New mutation causing hereditary hepatitis in the laboratory rat. J. Hered., 78,361-365.

Masuda, R., Yoshida, M.G., Sasaki, M., Dempo, K. and Man, M. (1988) Hereditary hepatitis in LEC rats is controlled by a singie autosomaf recessive gene. Lab. Anim., 22,166-169. Oyamada, M., Dempo, K., Fujimoto, Y., Takahashi, H., Satoh, MI., Mori, M., Masuda, R., Yoshida, M.G., Satoh, K. and Sato, K. (1988) Spontaneous occurrence of placental glutathione S-transferase-positive foci in the livers of LEC rats. Jpn. J. Cancer Res. (Gann), 79,5-8. Masuda, R., Yoshida, M.G., Sasaki, M., Dempo, K. and Man, M. (1988) High susceptibility to hepatocellular carcinomas in LEC rats with hereditary hepatitis. Jpn. J. Cancer Res. (Gann), 79,828-835. Enomoto, K., Takahashi, H., Sawaki, M., Sawada, N., Ikeda. T., Hattori, A. and Mori, M. (1989) High incidence of spontaneous hepatocellular carcinoma in a new mutant rat of hereditary hepatitis. Proc. Am. Assoc. Cancer Res., 30,194. Solt, D. and Farber, E. (1976) New principle for the analysis of chemical carcinogenesis. Nature, 263, 701703. Takahashi, H., Oyamada, M., Fujimoto, Y., Satoh, MA., Hattori, A., Dempo, K., Marl, M., Tanaka, T., Watabe, H., Masuda, R. and Yoshida, M.G. (19881 Elevation of serum alpha-fetoprotein of oval cells in the livers of LEC rats. Jpn. J. Cancer Res. (Gann), 79.821-827. 9 Tsuda, H., Lee, G. and Farber, E. (1980) Induction of resistant hepatocytes as a new principle for possible shortterm in vivo test for carcinogens. Cancer Res., 40, 11571164. 10 Sato, K. (1988) Glutathione S-transferase and hepatocarcinogenesis. Jpn. J. Cancer Res. (Gann), 79, 556-572. 11 Sugiyama, T., Takeichi, N., Kobayashi, H., Yoshida, M.C., Sasaki, M. and Taniguchi, N. (1988) Metabolic predisposition of novel mutant (LEC rats) to hereditary hepatitis and hepatoma: alterations of the drug metabolizing enzymes. Carcinogenesis, 9, 1569- 1572. 12 Doll, R. (1977) Strategy of cancer hazards to man. Nature, 265,589-596.