Znt. J . Cancer: 29, 333-336 (1882)

INDUCTION OF y-GLUTAMYLTRANSPEPTIDASE-POSITIVE ALTERED HEPATOCYTIC LESIONS BY COMBINATION OF TRANSPLACENTAL-INITIATION AND POSTNATAL-SELECTION Katsuhiro OGAWA, Kinya YOKOKAWA, Takashi TOMOYORI and Tamenori ONOB Department of Pathology, Sapporo Medical College, Sapporo, Japan 060.

The purpose of this study was to induce in postnatal life the selective growth of altered hepatocytic populations which had been induced in utero by various chemicals. Pregnant rats of a Wistar strain were given a single dose of various chemical carcinogens on the 18th gestational day. From two months postpartum, both the mothers and offspring were given a diet containing 2-acetylamino- fluorene (2-AAF) at a concentration of 0.02% for two weeks. Then, at the end of the first week after the 2-AAF feeding, a two-third partial hepatectomy was performed. All the animals were killed one week after the partial hepatectomy and were examined for the incidence of 7- glutamyltranspeptidase (GGT)-positive altered hepatocy- tic foci in the liver. The transplacental administration of not only a hepatic carcinogen, diethylnitrosamine (DEN) but also 7,12-dimethylbenzanthracene (DMBA), which does not usually produce hepatocellular carcinomas in adult rats, induced GGT-positive altered hepatocytes in the liver of the offspring. These hepatocytes grew into grossly visible hyperplastic nodules within a week after the two-third partial hepatectomy. The possible applica- bility of the transplacental initiation-postnatal selection model for the short-term assay of transplacental car- cinogenicity is discussed.

It is well documented that the exposure of pregnant animals to various carcinogens results in the occur- rence of tumors in the progeny. Most transplacental carcinogenesis in rats or other rodents had been cre- ated by giving single, multiple or continuous doses of chemicals to pregnant animals, maintaining the prog- eny in a normal condition and then examining the tumors arising in the progeny during a period of one or two years, or during the whole life span (Tornatis and Mohr, 1973; Rice, 1979; Mohr et al., 1980).

Recently, several investigators (Bulay and Watten- berg, 1970; Goerttler and Loehrke, 1976; Armuth and Berenblum, 1977) have observed a higher incidence and earlier appearance of tumors in experiments in which they have given carcinogens to pregnant mice and then have treated the progeny with the tumor promotors, phorbol or 12-0-tetradecanoyl-phorbol- 13-acetate. The results of their studies indicated that transplacentally-initiated cells respond to the tumor promotor in postnatal life.

The purpose of this study was to test whether the transplacental initiation-postnatal selection model is feasible in liver carcinogenesis. Various chemical car- cinogens were therefore administered to pregnant rats after which carcinogen-altered hepatocytes were selected according to the method of Solt and Farber (1976), which includes two weeks of dietary 2-AAF plus a two-third partial hepatectomy. By means of this procedure, it was possible to induce in the rats ex- posed to DEN or DMBA within the uterus hyperplas- tic hepatic nodules simiiar to those induced by con- tinuous feeding of hepatic carcinogens.

MATERIAL AND METHODS

Animals Female Wistar rats were mated and the dates were

recorded. On the 18th gestational day, the experhen- tal animals were given a single dose of various chemi- cal carcinogens, while the control rats received phy- siological saline. After the injection, daily inspection of the animals was camed out with the date of delivery and number of progeny born being recorded. Chemicals

The chemicals, number of doses and the routes of injection are illustrated in Table I. DEN (Tokyo Kasei, Tokyo, Japan), thioacetamide (TAA) (Merk, Darmstadt, Germany), urethan (UT) (Kishida, Osa- ka, Japan) were all dissolved in physiological saline at a concentration of 100 mg/d . DMBA (Sigma, Los Angeles, CA) was dissolved in corn oil (10 mg/ml) for intraperitoneal injection, or was suspended and soni- cated in normal rat serum (10 mg/ml) for intravenous administration. Dietary regimen for selection of altered hepatocytes

Offspring were weaned on the 21st postnatal day and were then fed a laboratory chow diet (Oriental Yeast, Tokyo, Japan) for 5 weeks. Thereafter, both the mothers and the offspring were given a diet con- taining 0.02% 2-AAF for h o weeks, after which a two-third partial hepatectomy was performed at the end of the first week of 2-AAF feeding according to the method of Solt and Farber (1976) (Fig. 1). All the rats were killed one week after the partial hepatec- tomy (at 10 weeks postpartum).

birth

carcinogen 1 weanling Dy killing

carcicogen pw killing -

(weeks postpartum) 0 3 8 9 10

gp::ibadfion suckling 0 ; y a l ;;YF FIGURE 1 - Schematic representation of the treatment of

the animals. (a) dietary regimen for progeny (b ) dietary regi- men for mothers.

Quantitation of GGT-positive hepatocytic lesions All the livers removed at the time of sacrifice were

sliced with a razor blade, with 3 or 4 slices of each right anterior, right posterior and caudate lobes being

period

Received: October 26, 1981.

334 OGAWA ET AL.

fixed in chilled acetone for GGT histochemistry (Oga- wa et al., 1980). The remaining tissue was fixed in Carnoy’s solution for H&E staining. Histochemical staining of GGT followed the method of Rutenberg et a1 (1969).

The areas of the histochemically stained sections were measured using an image analyzer, Digiplan (Kontron, Munich, Germany), and the number of GGT-positive hepatocytic lesions were counted by di- rect microscopic observation. The incidence of lesions was expressed in terms of the number per square cen- timeter of the sections. For each animal, 5 to 7 square centimeters of the sections were examined.

RESULTS

The number of progeny born and surviving at the end of the experiment, and body weight 9 weeks after birth are illustrated in Table I. Although the mortality of the offspring treated with DEN or DMBA in utero was higher than that of the control, the increase in body weight by the 9th week was almost the same as that of the control. UT and TAA did not appear to have any singificant effect on the mortality and growth of the offspring.

The livers of the mothers and progeny treated with the carcinogens appeared grossly normal at the time of partial hepatectomy , whereas histochemical examina- tion of the removed liver lobes revealed occasional small collections of GGT-positive hepatocytes (Fig.

Za). These changes were particularly observed in the offspring treated with DEN or DMBA.

The number of lesions per square centimeter of the sections one week after the partial hepatectomy is shown in Table I. Transplacental administration of DEN or DMBA clearly caused a significant increase in the number of the GGT-positive focal lesions (Fig. 2b) as compared to the control rats. DEN induced the same lesions in the liver of the mother, though their incidence was much lower than that in the progeny. There was no significant difference in the incidence of lesions between male and female offspring. DMBA resulted in a distinct increase in the lesions in the offspring especially when given to the mother in- travenously. When given intraperitoneally, it caused the induction of only small number of the lesions in the offspring. There was a tendency for U T and TAA, when given to the mothers intravenously, to slightly increase the number of the lesions in the offspring, but this was not statistically significant.

The morphological and histochemical properties of the focal lesions were basically the same as those de- scribed in previous literature (Gossner and Friedrich- Freksa, 1964; Friedrich-Freksa et al., 1969; Kitagawa, 1971; Kalengayi et al., 1975; Farber, 1976; Scherer and Emmelot, 1976; Pitot et al., 1978).

Organs other than the liver appeared grossly normal and no pathological or teratological changes were found in the progeny.

FIGURE 2 - Livers of progeny whose mothers were given DEN (100 mg/kg body weight) on gestation day 18. (a) Small collection of GGT-positive hepatocytes seen at 9 weeks postpartum in a liver removed at the time of two-third partial hepatectomy. X 100. (b ) GGT-positive hyperplastic hepatic nodules one week following a two-third partial hepatectomy. X 13.

TRANSPLACENTAL HEPATIC CARCINOGENESIS 335

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DISCUSSION

The combination of transplacental administration of carcinogens and the postnatal dietary regimen for selection of carcinogen-induced altered hepatocytes resulted in a marked increase in the number of GGT- positive focal hepatocytic lesions in the livers of prog- eny, indicating that the transplacental initiation-post- natal selection model is feasible in liver carci- nogenesis. While the dietary regimen (two weeks of 0.02% dietary 2-AAF plus a two-third partial hepatectomy) without in utero carcinogenic treatment induced a small number of the lesions, their incidence was usually less than 0.5/cm2 of the sections. It is probable that altered hepatocytes induced during in- tra-uterine life, which were seen as occasional small collections of GGT-positive hepatocytes prior to the partial hepatectomy, responded to the dietary regi- men, resulting in the formation of these nodules.

The incidence of the lesions in the offspring was higher than that in the mothers especially when DMBA, which is not a classical hepatocarcinogen, was given. This is compatible with the findings that chemicals which are not usually hepatocarcinogenic or have a weak hepatocarcinogenicity initiate hepatocel- lular carcinomas and hyperplastic hepatic nodules when given during fetal or newborn periods or periods of restorative proliferation after partial hepatectomy or hepatic necrosis (reviewed by Craddock, 1976). There is cumulative evidence that proliferating hepa- tocytes, especially in the S phase, show an increased sensitivity to the initiating effect of carcinogens, pre- sumably due to the fact that replication of nuclear DNA may be important for fixation of altered cells on initiated state (Pound, 1968; Chernozemski and War- wick, 1970; Craddock, 1976; Kaufman e l d., 1978; Cayama et al., 1978).

On the other hand, it has been shown that the metabolism of carcinogens in perinatal and restora- tively proliferating livers is different from that of the adult liver, that is, carcinogens stay within the hepatic tissue over a longer period as compared to the normal liver, which may be related to the increased sensitivity to carcinogens (Craddock, 1976). The mechanism of action of transplacentally administered carcinogens, such as DEN and DMBA, both of which need metabolic activation through microsomal drug-meta- bolizing enzymes, however, seems more complicated since the enzyme system is rather poorly developed in the fetal liver. It is possible that the carcinogens acti- vated in the maternal liver and placental tissue, o r those excreted into milk, may play some role in induc- tion of the hepatic lesions.

Another factor which should be taken into consider- ation is the fact that the effects of the 2-AAF used as the selecting agent in the present study were signifi- cantly variable depending on the age and sex of the animals. That is, the lower incidence of the lesions in the mothers may partly be due to a weaker selection pressure as compared to the offspring. Inhibition of the liver weight after partial hepatectomy by 2-AAF feeding, however, was of almost the same degree in both mothers and offspring in the present study, sug- gesting that the difference in the incidence of the hepatic lesions between the mothers and offspring

336 OGAWA ET AL.

may not be dependent on the difference in the selec- tion pressure.

The injection route seems to be of significant im- portance in the initiating action of the carcinogens for the fetal liver. Intraperitoneal injection of DMBA dis- solved in corn oil exhibited only a slight effect in fetal liver in the present study. The carcinogens may be metabolized and detoxicated by the maternal liver and may not reach the fetal liver tissue.

effect was assayed by the incidence of carcinogen-al- tered hepatocytic lesions which arose much higher incidence than overt hepatocellular carcinomas. Al- though the present experimental model may not be a substitute for the classical model, the shortness of the experimental period and the high incidence of focal lesions seems to indicate its possible effectiveness as a short-term screening model for transplacental car- cinogenicity.

The characteristics of the transplacental initiation- ACKNOWLEDGEMENTS

postnatal selection model in the present study were (1) This work was supported by grants from the Minis- that total experimental period was much shorter as ' try of Education, Science and Culture, Japan, from compared to that in the classical transplacental car- the Hokkaido Society for Promotion of Gerontology cinogenesis experiments, and (2) that the carcinogenic and from the Japanese Society of Basic Oncology.

REFERENCES

ARMUTH, V., and BERENBLUM, I., Possible two stage transpla- aflatoxin B-induced carcinogenesis. I . Nat. Cancer Inst., 55,579- cental liver carcinogenesis in C57BU6 mice. fnt, 1. Cancer, 20, 588 (1975). 292-295 (1977). KAUMAN, D.G., KAUFMAN, W.K., RICE, J.M., and WENK, BULAY, O.M., and WATTENBERG, L.W., Carcinogenic effects of M.L., Cell cycle variation in susceptibility to hepatocarcino- subcutaneous administration of benzo(a)pyrene during pregnan- genesis by MNU. Proc. A m . Ass. Cancer Res., 19, 183 (1978).

CAYAM& E., TsuDA* T.i SARMA, D.S.R., and E., hi- and hepatomas of the liver of rats fed 2-fluorenylacetamide. tiation of chemical carcinogenesis requires cell proliferation. can,,, 62, 207-216 (1971). Nature (Lond.), 275,60-62 (1978). CHERNOZEMSKI, I.N., and WARWICK, G.P., Liver regeneration ~ ~ ~ ; ~ ; ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ e ~ ~ ~ z ~ ~ ~ ~ ~ ~ ~ ( ~ ~ ~ ~ ~ ~ and induction of hepatomas in B6AF1 mice by urethan. Cancer Res., 30,2685-2690 (1970). OGAWA, K., SOLT, D.B., and FARBER, E., Phenotypic diversity

cancer, In,.' M.M. cameron, D,A, ~ i ~ ~ ~ l l and G,p, Warwick tions in liver carcinogenesis. Cancer Res., 40, 725-733 (1980).

On the progeny. exp' Biol. Med', 13', 84-86 (1970)' K ~ A G A W ~ , T, , Histochemical analysis of hyperplastic lesions

CRADDOCK V.M., Cell proliferation and experimental liver as an early property Of putative preneoplastic hepatocyte

(ed.), Liver ceN cuncer, pp. 153-201, Elsevier, Amsterdam, New York, Oxford (1976). FARBER, E., Hyperplastic areas, hyperplastic nodules and hyper- basophilic areas as putative precursor lesions. Cancer Res., 36,

FRIEDRICH-FREKSA, H., PAPADOPULU, G., and GOSSNER, W., Histochemische Untersuchungen der Cancerogenese in der Rat- tenleber nach zeitlich begrenzter Verabfolgung von diathylni- trosamin. 2. Krebsforsch., 72, 240-253 (1969). GOERTIZER, K., and LOEHRKE, H., Diaplacental carcinogenesis: Initiation with the carcinogens dirnethylbenzanthracene (DMBA) and urethane during fetal life and postnatal promotion with phorbol ester "PA in a modified 2-stage BerenblumNa- thram experiment. Virchows Arch. A, Path. Anat. Histol., 372,

G~SSNER, W., and FRIEDRICH-FREKSA, H., Histochemische Un- tersuchungen iiber die Glucose-6-Phosphatase in der Rattenleber wahrend der Kanzerisierung durch Nitrosamine. 2. Naturforsch.

KALENGAYI, M.M.R., RONCHI, G., and DESMET, V.J., Histo- chemistry of gamma glutarnyl transpeptidase in rat liver during

2532-2533 (1976).

2-38 (1976).

(B), 19, 862-863 (1964).

PITOT, H.C., HARSNESS, L., GOLDWORTHY, T., and KITAGAWA, T., Biochemical characterisation of stages of hepatocarcino- genesis after a single dose of diethylnitrosamine. Nature (Lond.), 271, 456-459 (1978). POUND, A.W., Carcinogenesis and cell proliferation. N.Z. Med. J., 67, 88-99 (1968). RICE, J. (ed.), PerinaruZcurcinogenesis. National Cancer Institute Monograph, Vol. 51 (1979). RUTENBERG, A.M., KIM, H., FISHBEIN, J.W., HANKER, J.B., WASSERKRUG, H.L., and SELIGMAN, A.M., Histochemical and ultrastructural demonstration of y-glutamyl transpeptidase activi- ty. J. Histochem. Cytochem., 17,517-526 (1969). SCHERER, E., and EMMELQT, P., Kinetics of induction and growth of enzyme-deficient islands involved in hepatocarcino- genesis. Cancer Res., 36, 2544-2554 (1976). SOLT, D., and FARBER, E., New principle for the analysis of chemical carcinogenesis. Nature (Lond.), 263, 701-703 (1976). T o m n s , L., and MOHR, U. (ed.), Tramplacental Carcino- genesis. IARC Scientific Publications No. 4, International Agen- cy for Research on Cancer, Lyon (1973).