Int. J . Cancer: 31,775-777 (1983)

INITIATION OF FOCAL HYPERPLASTIC HEPATIC LESIONS BY TRANSPLACENTAL ADMINISTRATION OF ETHYLNITROSOUREA IN RATS OF F1 GENERATION, AND NO TRANSMISSION OF THE EFFECT TO F2 AND F, GENERATIONS Katsuhiro OGAWA, Kinya YOKOKAWA, Takashi TOMOYORI and Masanobu NARASAKI Department of Pathology, Sapporo Medical College, S . l , W.17, Chuo-ku, Sapporo, Japan 060.

Administration of ethylnitrosourea (ENU) to pregnant rats in the late stage of gestation has been known to have a mainly neurocarcinogenic action on the progeny. How- ever, when transplacental administration of ENU was combined in postnatal life with a brief dietary exposure to 2-acetylaminofluorene (2-AAF) and a two-thirds partial hepatectomy, numerous focal hyperplastic lesions of y-glutamyltranspeptidase (GGT)-positive hepatocytes emerged in the liver of the progeny. On the other hand, rats of F, generation born of F, parents and F, rats born of F2 parents showed no increase in the incidence of hepatic lesions after treatment with the same dietary regimen. It was suggested that direct interaction between fetal liver cells and the carcinogen is important for Occurrence of initiated hepatocytes and that the initiation effect on rat liver by ENU does not persist in the descendants of F, and F, generations.

ENU is well known to induce various neoplasms in progeny when given to pregnant animals. The inci- dence of tumors and the organs involved are depen- dent on the species, strains and developmental stages of the animals (reviewed by Mohr et al., 1980). In the case of rats, transplacentally-administered ENU dur- ing the late stage of pregnancy is mainly carcinogenic to the nervous tissues, and, to a lesser extent, to kidney, hematopoietic tissues, etc. (Ivankovic and Druckrey, 1968; Wechsler et al., 1969; Druckrey etal., 1970; Swenberg ef al., 1972). Although the liver is one of the major target organs in mice, it is involved to a much lesser extent in rats (Rice, 1973; Vesslinovitch, 1973; Schmahl and Kriegel, 1978). ENU is also known as a direct-acting carcinogen not requiring metabolic activation in viva for carcinogenic activity, and it pre- sumably acts systemically on the fetus when given transplacentally. We previously demonstrated that the combination of transplacental administration of vari- ous carcinogens and postnatal selection of carcinogen- altered hepatocytes resulted in the emergence of focal GGT-positive hepatocytic lesions (Ogawa et al., 1982). In this study, we gave ENU to pregnant rats and promoted in postnatal life the growth of the ini- tiated cells in the liver by a brief dietary exposure to 2-AAF and a two-thirds partial hepatectomy .

There have been several reports stating that the car- cinogenic effects of ENU and of other carcinogens which act transplacentally persist in the descendants of the second and even the third generation (Shay et al., 1952; Gelstein, 1961; Tanaka, 1973; Nomura, 1975; Tomatis et al., 1977; Tomatis, 1979; Rao, 1982). Therefore, we also determined the incidence of hepa- tic lesions in the F2 and F, descendants of F, rats exposed to ENU in utero.

MATERIAL AND METHODS

Animals and treatment Fischer 344 (F344) rats were obtained from Charles

River Co., Azugi. Male and female rats were mated and the first day of pregnancy was defined as the day on which a vaginal plug was observed. Pregnant rats received a single dose of ENU on day 18, 19 or 20 of gestation. ENU was dissolved in 0.01 M citrate buffer PH 4.0 at a concentration of 25 mg/ml and injected into the femoral vein of the rats at a dose of 40, 80 or 100 mg/kg body weight. Controls were given the buffer alone. Thereafter, the animals were inspected every day, and the date of delivery and the number of progeny were recorded.

All progeny were weaned on the 21st day after birth and given the laboratory chow diet (Oriental Yeast Co., Tokyo) for 5 weeks. Then one group of siblings from each family was used for selection of altered hepatocytes, and the group was used for reproduction of the F, generation. Selection of altered hepatocytes

When the progeny reached 8 weeks of age, the mothers and one group of F1 rats were given a diet containing 0.02% 2-AAF for 2 weeks. A two-thirds partial hepatectomy was carried out on these rats 1 week after the initiation of the 2-AAF diet (Solt and Farber, 1976). All rats were killed one week after par- tial hepatectomy. F2 and F3 generations

To obtain rats of F2 generation, one group of rats, consisting of one female and one male, were paired at the age of 12 weeks. Rats of the F3 generation were obtained by mating the F2 rats. A11 the rats were weaned on the 21st day after birth and given a chow diet for 5 weeks. F, and F3 descendants and their parents were treated with the regimen for selection of the hepatic lesions from 8 weeks after birth. Quantitation of y-glutamyltranspeptidase positive he- patocytic lesions

The livers were sliced and fixed in chilled acetone for GGT histochemistry (Ogawa et al., 1980). His- tochemical staining of GGT activity followed the method of Rutenberg et al. (1969). The areas of the histochemically-stained sections were measured using an image analyzer (Digiplan, Kontron, Munich, Ger- many), and the number of GGT-positive areas was counted microscopically as described elsewhere (Oga- wa et al., 1982). The incidence of lesions was ex-

Received: January 25, 1983 and in revised form March 29, 1983.

776 OGAWA ET AL

TABLE I - INCIDENCE OF HEPATOCYTIC LESIONS AND OTHER NEOPLASMS IN F, RATS TREATED WITH 2-AAF PLUS A TWO-THIRDS PARTIAL HEPATECTOMY

Other neoplasms Hepatocytic lesions5 Treatment Dose' Gest'4 Progeny ~ Mother F, rats day born

Mother Male Female N8 Non-NY ThatLD N non-N Tirat

Controll - ENU2 40

40 80 80

100 100 100 100

18 8 0.0 18 8 1.1 21 8 0.0 18 2 1 .o 21 4 0.3 18 6 3.0 18 5 2.7 19 6 died 20 9 6.8

O.l-tO.1 (2)6 0.2 (2) 0.5rtO.3' (2) 0.5+0.6 (5) 3.2-tO.6' (5) 1.9+1.0 (3) 2.8k0.3' (2) -

3.0 (1) 3.7k0.4 (3) 7.5 (1) 6.6-tl.5 (2)

9.0-tl.O (3) -

8.3k2.6 (4) - 4.2f1.6 (2) 5.8 (1)

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 0 0 (4) 0 0 0 (7)

0 0 0 (2) 0 0 0 (4) 2 S ' I 1.0 (3) 0 0 0 (3) 0 0 0 (3) 2 0 0.5 (4)

0 0 0 (8)

I A control rat was given 0.01 M citrate buffer on day 18 of gestation. When the progeny reached 8 weeks of age, either the mother or the progeny was treated the dietary regimen for selection of the hepatic lesions. - 'Pregnant rats (at day 18-21 of gestation) were treated with ENU (40-100 mgikg body weight) dissolved in the citrate buffer. Mothers and offspring were treated with the selection regime as above. -'mgikg body weight. - lDar of gestation on which ENU was injected. - 5Mean number of lesions per cm' of section -C SD. - 6Number in parentheses is the number of animals. - Difference is significant (p <0.01) compared to controls. - 8Neurogenic tumors. - 'Grossly visible neoplasms other than neurogenic tumors. - 'OMean number of tumors per rat. - "Salivary gland tumor. One small letter represents one tumor.

pressed as the number per cm2 of the sections. For each rat, 5 to 7 cm2 of sections were examined. Examination of organs other than liver

All rats, including those which died during the course of the experiments, were autopsied. The tis- sues were fixed in Carnoy's solution and investigated for the presence of neoplastic changes. Urinary blad- ders were expanded by injection of a small volume of fixative before removal for inspection of luminal sur- faces.

RESULTS

Incidence of GGT-positive hepatocytic lesions and other neoplasm in F, rats

The livers of rats exposed to ENU in utero and given the regime for selection of hepatic lesions post- natally showed a significant increase in the number of GGT-positive foci of altered hepatocytes as compared to the control rats given citrate buffer in utero and treated with the selection regime postnatally. The inci- dence of the lesions (Table I) was roughly proportion- al to the doses of ENU. When 100 mg ENU/kg body weight were given between 18 and 20 days following the initiation of pregnancy, the incidence of hepatic lesions was almost the same in each family. The fre- quency of hepatic lesions in the livers of the mothers was lower than in their progeny, whereas there was no significant difference between males and females.

The progeny showed occasional neoplastic changes in the organs other than the liver at the age of 10

weeks, when the experiment was concluded (Table I). Four gliomas were observed in the cerebrum or spinal cord in 4 out of 14 rats born of a mother treated with 100 mg ENU/kg body weight. Besides these neurogenic tumors, one cervical tumors resembling Warthin's tumor of the salivary gland, two micro- scopic lung adenomas and one focal dysplasia of pan- creatic acinar cells were detected.

Incidence of hepatic lesions in F2 and F3 generations F2 rats born of F, parents and F3 rats born of F,

parents were treated with the regime for the selection of hepatic lesions 8 weeks after birth. The incidence of hepatic lesions is listed in Table 11. These rats did not show a significant increase in the number of hepatic lesions as compared to control rats. Furthermore, we found no neoplastic change in these rats on autopsy at 10 weeks of age, at which time experiments were con- cluded.

DISCUSSION

It has been shown that, when the progeny of mothers which had been given chemical carcinogens or radioisotopes was treated with TPA or croton oil in postnatal life, there was a remarkable increase in the incidence of tumors not only in their skin but also in the internal organs (Bulay and Wattenberg, 1970; Goerttler and Loehrke, 1976; Armuth and Beren- blum, 1977). Using the same experimental design, we previously demonstrated that induction of GGT-posi- tive hepatocytic nodules was feasible in rats which had been exposed to carcinogens in utero by a regime con-

TABLE I1 - INCIDENCE OF GGT-POSITIVE HEPATOCYTIC LESIONS IN F, AND F, RATS

F, F, F, F1 Treatment Dose) Gest'4

day Father Mother Male Female Father Mother Male Female

Control' - 18 died 0 0.4 (1)6 0.1+0.1 (4) died 0 0 (2) O . l f O . l (4) ENU2 100 19 4.05 3.1 0.1+0.1 ( 5 ) O.lkO.1 ( 5 ) died 0.1 - 0.1+.0.1 (2)

100 20 4.4 4.6 0.1+0.1 (3) 0 (4) 0.5 0.2 O.lrt0.2 (7) 0.2k0.1 ( 5 )

control rat was given 0.01 M citrate buffer on day 18 of gestation. One male and one female of the progeny were mated 12 weeks after birth for reproduction of F, generation. When F, rats reached 8 weeks of age, the F, parents and F, rats were treated with the dietary regimen for selection of the hepatic lesions. F, rats were produced from the F, rats. The F2 parents and F, descendants were also treated with the selection regime when F, rats reached 8 weeks of age. - ,ENU dissolved in the citrate buffer was given to pregnant rats. The F, and F, descendants were produced by brother-sister mating and treated in the same way as described above. - )mg/kg body weight. -' Day of gestation on which ENU was injected. -'Number of lesions per cm2 of sections ? SD. ~ 'Number in parentheses represents the number of animals.

sisting of 2 weeks of dietary exposure to 2-AAF and a two-thirds partial hepatectomy (Ogawa et al., 1982). This model enabled us to quantitate the transplacental initiation effect of various carcinogens on fetal liver within a relatively short time by measuring the inci- dence of GGT-positive foci. Using the same model, the present study showed that ENU is also a potent initiatior of altered hepatocytes in fetal rat liver.

Transplacentally-administered ENU in rats is known to be a potent neurocarcinogen, whereas it rarely induces hepatic tumors (Ivankovic and Druck- rey, 1968; Druckrey etal., 1970; Swenberg et al., 1972; Mohr et al., 1980). The results of this study indicated that, although ENU induces a significant number of altered hepatocytes in fetal liver, they may not prog- rtss further in the carcinogenic process, remaining la- tent without promotion.

There have been number of reports of transplacen- tal administration of carcinogens elevating the inci- dence of neoplasms in the second and third genera- tions (Shay et al., 1952; Gelstein, 1961; Tanaka, 1973; Nomura, 1975; Tomatis et al., 1977; Tomatis, 1979: Rao, 1982). Tomatis et al. (1977) reported that when

F344 rats were given ENU during the late stage of gestation, the incidence of neurogenic tumors in- creased not only in the F, generation but also in F2 and F3 generations born of F, parents. The mecha- nism of such carcinogenesis is considered to be me- diated by some alterations in germ cells (Tomatis, 1979). There are also several reports that treatment of germ celIs by X ray or chemical carcinogens results in neoplasms and spot mutation in descendants (Russel et al., 1974; Tomatis et al., 1981; Nomura, 1982). However, in the present study, we did not demon- strate an increase in the incidence of hepatic lesions in the F, and F3 progeny derived from F, rats exposed to ENU in utero. Therefore, we assumed that a direct interaction between the carcinogen and fetal liver cells is important for the occurrence of altered hepatocytes, and that it is unlikely that the initiation effect of ENU on rat liver persists through several generations.

ACKNOWLEDGEMENTS

This work was supported by grants from the Hok- kaido Society for the Promotion for Gerontology and from the Japanese Society of Basic Oncolocy.

' TRANSPLACENTAL INITIATION BY ENU 777

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