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A Histological Study of the Promotive Effect of Diethylstilbestrerol on Diethylnitrosamine Initiated Carcinogenesis of Liver in Rat
Path. Res. Pract. 178,339-344 (1984)
A Histological Study of the Promotive Effect of Diethylstilbestrerol on Diethylnitrosamine Initiated Carcinogenesis of Liver in Rat Ding Lian, Zou Daoyun, Chen Jinyan, Wang Juantan, Wang Yuanbu, Pan Shaoru and Wand Shuhua Department of Pathology, Institute of BasicMedicalSciences, Chinese Academy of Medical Sciences, Beijing, PR China
SUMMARY
A single dose (80 mg/kg) of diethylnitrosamine (DEN) wasgiven orally to castrated female Wistar rats. One week after that one half of the animals were treated with diethylstilbestrol(DES) J mg/kg/once a week subcutaneously. The other half of the animals received no any hormone or hormone derivatives. The change of the liver cells in animals treated with DEN alone failed to progress beyond the stage of hepatocellular alterations in foci or neoplastic nodules ioithm 8 months, while most of those antmals which receiued DES treatment after DEN initiation developedhepatocellular carcinomas after 6 months. This result denotes that the DES exerts a definite promotive effect on DEN initiated liver cell carcinogenesis.
The synthetic estrogens such as ethinyl estradiol and DES have been reported to provide a cocarcinogenic or promotive effect on liver tumorigenesis in some species of animals. Cameron et a1. demon strated that the ethinyl estradiol was a promoter of precancerous liver lesions in male Fischer 344 rats'. Sumi et al. in their experiments showed that the DES played a synergistic effect with Nbutyl-N-nitrosourea or fast neutron (s) irradiation on hepatoma development in castrated male WF rats", We had preliminarily reported that DES provided a promotive effect on liver carcinogenesis in castrated female Wistar rats being formerly initiated by DEN 3• In this experiment the histological changes regarding to the promotive effect of DES in liver carcinogenesis were further studied.
Materials and Methods Seventy two female Wistar rats, 120-150 gms in weight, recieved a single dose of DEN orally (80 mg/kg) one week after castration. Ten rats were sacrificed 3 and 7 days after DEN © 1984 by Gustav FischerVerlag,Stuttgart
administration respectively (5 rats for each) for studying the toxic effect of DEN and the repairing process taking place in the livers. One week after DEN administration, the rats were divided into 2 groups at random. In the first group (DD group) DES was injected subcutaneously in all 31 rats, 3 rng/kg/once a week. In
the second group (D group), 31 rats were used as controls . By the end of the 2nd week as well as the first, 2nd, 4th, 6th and 8th months after DEN, 5 or 7 rats of each group were sacrificed after a fast of 15-17 hours (over night) respectively. Liver tissue blocks were taken from each rat for the following examinations.
1. Routine histopathological study: Tissue blocks, 3 mm in thickness, taken from each lobe of the liver were fixed in 10% formalin for 24 hours. Paraffin sections were stained with hematoxylin and eosin. The histological classification of the hepatic lesions was described in accordance with the nomenclature suggested by The Institute of Laboratory Animal Resources generally used for typing of precancerous and cancerous lesions experimentally induced in rat's liver. 2. Glycogen stain: Tissue blocks taken from left lobe of the livers were fixed and dehydrated in absolute alcohol. The paraffin sections were stained with Best's carmine. 0344-0338/84/0178-0339$3.50/0
340 . Ding Lian, Zou Daoyun, Chen jinyan, Wang juantan, Wang Yuanbu, Pan Shaoru and Wand Shuhua
3. Histochemical demonstration of gamma-glutamyl transferase (GGT) in tissue blocks: Tissue blocks of left and left-middle lobes of liver were taken 4, 6 and 8 months after DEN adminis-
tration. Blocks of 2 mm in thickness, after being rinsed with normal saline (N. S.) and immersed in 0.1 M Tris-HCl buffered saline (pH 7.4) for 5 minutes were incubated in GGT substrate for 1 hour in room temperature. The blocks were rinsed again with N.S.; immersed in 0.1 M copper sulfate solution for 5-10 minutes; then washed with N. S. and were examined under stereomicroscope.
Substate for GGT contained': Garnma-Lglutamyl-c-naphthylamine 5 mg dissolved in 4 ml N.S. Fast blue B 5 mg dissolved in 1 ml N. S. Glycylglycine 5 mg dissolved in 1 ml N. S. 0.1 M Tris-HCl buffered saline, ph 7.4 4 ml. Mixed and filtered immediately before use
4. Histochemical demonstration of GGT and ornithine carbamyltransferase (OCTt· 7: Cryostat sections (7.5-10 11 in thickness) of left middle lobe of liver were fixed in -79 °C. acetone over night. Procedure for GGT was similar as those for tissue block staining. Procedure of histochemical demonstration of OCT: Sections, after fixation, were rinsed with 0.05 M Tris-maleate buffer solution (pH 7.3), and incubated in OCT substrate for 30-35 minutes in room temperature. Substrate for OCT contained: Carbamyl phosphate Li-salt L-ornithine
5 mg 5 mg
0.05 M Tris-maleate buffer solution, pH 7.3 4.5 ml Sucrose 0.8 g Distilled water 5 ml After well mixed 0.5 ml of 2% lead nitrate was added drop by drop under vigerous stirring and then filtered. Substrate must be freshly prepared before use. After that, sections were rinsed with distilled water and immersed in 1% ammonium sulphite for 1 minute. Rinsed the sections with distilled water and stained with 0.5% methyl green if desired and mounted either with glycerin-jelly or dehydrated with alcohol and then mounted with Canada Balsam after xylol for clearing.
Results Three days after DEN administration, the livers slightly swelled to about 3.2% of the body weight (Table 1, normal values: 2.5-3.0%). There was moderate congestion being dark red in colour, and over the cutting surfaces the outline of lobules could still be identified. Microscopically, central and bridging necrosis of liver cells were obtained in nearly all of the lobules. Liver cells over the peripheral zone of the lobules were moderately hyperplastic with mild basophilic cytoplasm, enlarged nuclei, prominent nucleoli and scattered mitotic figures. Mild lymphocytic infiltration presented over the portal spaces but small biliary ductules intacted. OCT activity disappeared in those necrotic liver cells (Fig. 1).
liver weight ) Table 1. Averagecomparative weight of livers ( x 100% in each group (the numbers of animals are quoted in body weight parentheses) Durations after DEN DO group ± S.D. D group ± S.D.
3 days
3.2 (5)
± 0.32
VI M
7days
3.8 (5)
± 1.12
3.8 (5) 3.5 (5)
1M
± 0.51 ± 0.25
4.8 (5) 3.0 (5)
4M
2M
± 1.02 ± 0.19
3.8 (7) 2.7 (5)
± 0.46 ± 0.37
3.2 (5) 2.5 (5)
8M
6M
± 0.13 ± 0.13
4.4 (5) 2.3 (5)
± 0.88 ± 0.27
4.6 (4) 2.3 (6)
± 1.98 ± 0.29
Fig. 1. 3 days after DEN, OCT activity vanished in central necrotic areas, OCT, x 100.
Diethylnitrosamine Initiated Carcinogenesis of Liver in Rat . 341
be easily distinguished from the GGT positive nodules or masses mentioned above. Microscopically, the cytoplasm of the periportal liver cells in 4 months DD group animals were moderately basophilic and their nuclei were deeply stained. Scattered mitotic figures (1-2/HP) might be seen in the midzonal regions as well as large- or multi-nucleated liver cells presented in the central portions of the lobules. Foci and areas of cellular alterations similar as those seen in animals of single DEN group were also obtained in the sections (Fig. 2). Neoplastic nodules began to develop in 2 of the 5 DD group animals 4 months after DEN and they grew up to form varying sized nodules in all of the DD animals 6 months after DEN administration (Fig. 3). In a few nodules the basophilic liver cells looked invading into the surrounding tissue which was considered to be a criteria of undergoing malignant change. Trabecular hepatocellular carcinomas were found in 4 out of 5 rats in DD group after 6 months of DEN intake and in 3 out of 4 animals after 8 months of DEN. These cancer cells arranged in cords, acini or masses and their cytoplasm revealed basophilic, nuclei deeply stained and nucleoli increased or enlarged. The mitotic figures were numerous in the cancer nests (4-5/HP) (Fig. 4). The borders of these cancer masses were ill-defined and Best's carmine stain revealed no apparent glycogen storage in most of the cancer cells. GGT reaction of these cancer cells showed strong positive (Fig. 5) while OCT activity was markedly decreased or vanished (Fig. 6). Cirrhosis of liver obtained in one DD rat of the 8th month group and OCT activity of the liver cells in cirrhotic nodules was positive.
Seven days after DEN administration, livers became moderately enlarged making up 3.8% of the body weight (Table 1). Microscopically, most of the central necrosis disappeared, only occasional necrotic or eosinophilic degenerated liver cells found surrounding the central veins. Numerous binucleated liver cells and scattered mitotic figures still presented in the peripheral and midzonal regions of the lobules. OCT activities repaired to normal range in most of the lobules. Within 2 months of DEN administration, livers of animals in both groups slightly or moderately enlarged (Table 1). Microscopically, all these livers revealed essentially normal. The OCT activities also showed normal. After 4 months of DEN administration, livers of those rats recieved DEN alone almostly recovered to normal in appearence grossly and GGT reactions showed no positive signs or only got occasional red spots in the gross specimens (Table 2). Microscopically, both liver cells and lobules were well preserved except more or less foci and areas of hepatocellular alterations seen (Table 2). Most of these foci consisted of clear and acidophilic liver cells while those foci composed of basophilic cells did not exceed 10% totally. Best's carmine stain showed marked storage of glycogen in the clear and acidophilic liver cells but OCT activities decreased or disappeared. Livers of rats in DD group were larger than the normal ones and progresively increased their weight until up to nearly twice the weight of the normals after 6 months of DEN administration (Table 1). Macroscopically, these livers showed dark red in colour and granular in appearence with dullrounded edges. The cutting surfaces were markedly bulging up with scattered gray-yellowish granules, nodules or masses. GGT reaction gave these granules, nodules or masses reddish in colour which would be easily recognized and counted under stereomicroscope. The number of the GGT positive granules and nodules of DD group animals were apparently more than those of the D group animals (Table 2). In some cases of DD group, the periportal liver tissue might also show a red colour in GGT reaction giving a nutmeg in appearence. These streak mottled areas could
Discussion DES have been used as an estrogen substitute clinically. Experimentally, it is known to have the abilities in inducing various tumors such as tumors of uterine cervix and kidneys in rodents. Formerly, Reuber et al.8 reported that DES provided a protective effect on N-2-fluorenyldiacetamide induced cirrhosis of liver and reduced the
Table 2. Hepatic lesions of the rats Months after DEN Groups Number of animals
2 DD 7
D 5
GGT (+) foci in gross specimen avo no.lcm 2 ± S.D. Foci of cellular alterations avo no.zcrrr' 0.8 ± S.D. 10.9 % of basophilic cell foci ± S.D.
0.7 0
4 DD 5
6 DD 5
D 5
8 DD 4
D 5
D 6
1.0 ±0.6
0
4.6 ±2.8
0.9 ±0.8
3.3 ± 2.2
0.9 ±0.5
1.0 ±0.4 25.5 ± 10.8
2.1 ±1.3 7.2 ±7.0
6.2 ±2.8 26.9 ± 11.2
2.3 ±1.4 4.0
4.7 ± 1.8 29.5 ± 11.4
3.5 ±2.3 7.8 ±6.7
Neoplastic nodule"
0
0
2
0
5
0
4
1
Liver cell cancer" *
0
0
0
0
4
0
3
0
* Number of rats developed neoplastic nodules ** Number of rats developed liver cell carcinomas
342 . Ding Lian, Zou Daoyun, Chen jin yan, Wang j uantan , Wang Yuanbu, Pan Shaoru and Wand Shuhua
Fig. 2. DD group, 6 month s after DEN, neoplastic nodule composed of vacuolated clear liver cells, HE, X 100.
Fig. 3. DD group , 6 months after DEN, neoplastic nodule composed 'o f basophilic liver cells, HE, X 100.
Fig. 4. DD group, 8 months after DEN, hepatocellular carcinoma and cirrhosis of liver, HE, X 100.
Diethylnitrosamine Initiated Carcinogenesis of Liver in Rat . 343
Fig. 5. DD group, 6 months after DEN, Liver cell cancer shows GGT positive reaction. GGT, X 100.
Fig. 6. DD group, 8 months after DEN, OCT activity markedly decreased in liver cell cancer. OGT, X 100. incidence of hepatoma in A X C male rats. Recently, works denoted that DES was a cocarcinogen of liver in castrated male rats and DES alone might induce a definite hepatocarcinogenicity in those rats even with only a low incidence of liver cells tumor', In this experiment, the livers after a single dose (80 mg/kg) of DEN failed to progress beyond lesions of altered cell foci and occasional neoplastic nodule within 8 months. If the animals were treated with DES persistently for 4 months after DEN initiation there would be marked hyperplasia of periportal liver cells and numerous basophilic altered cell foci present in livers and neoplastic nodules began to develop in these animals. If DES giving was lasted for more than 6 months, not only all the animals developed neoplastic nodules but also hepatocellular cancers formed in most of these animals. The results denote that DES exerts a promotive effect on DEN initiated liver carcinogenesis in castrated female Wistar rats.
DES is a synthetic analogue of estrogen. Li et al." recently reported that a high incidence (70%) of hepatoma in castrated male Syrian Hamsters were obtained after treatment with DES plus a-naphthoflavone (a-NF), an inhibitor of microsomal mixed function oxidase. Although the estrogen receptors of these liver cells elevated up to about 5-fold, anyhow, no difference was obtained in estrogen receptor levels in comparing with those found in other species of animals. Therefore the authors considered that the induction of hepatoma by DES plus a-NF in these hamsters was possibly due to a non-hormonal mechanism. Sumi et al.2 considered that the DES might be a weak carcinogen or might exert as a promotor indirectly by its induction of hypersecretion of pituitary hormone such as the secreta of mammosomatotropic pituitary tumor which have been shown to be a promoter of chemical carcinogen induced hepatocarcinogenesis. Whether the promotive role of DES on hepatocarcinogenesis is due to its chemical
344 . Ding Lian, Zou Daoyun, Chen ]inyan, Wang ]uantan, Wang Yuanbu, Pan Shaoru and Wand Shuhua character (as a weak carcinogen) or due to its hormonal effect (enhancement of protein synthesis or acceleration of hyperplasia of liver cells) or owing to both still remains to be studied. GGT reaction in tissue blocks shows some altered liver cell foci, neoplastic hepatocellular nodules and cancer masses in red colour and makes these lesions easily be recognized by naked eye or under stereomicroscope particularly if these are only a few small nodules presented on the cutting surfaces. The OCT activities of immature or embryonal liver cells are very weak or even negative. The precancerous and cancerous hepatocytes also reveal lowered or vanished OCT activity. Sometimes the degenerated or necrotic liver cells may also give a weak or vanished OCT reaction, but the positive GGT reaction only presents in proliferating liver cells. Therefore it seems better to evaluate the OCT negative lesions accompanying with the result of GGT reaction for making a more definite decision of proliferating immature liver cells lesions. References 1 Cameron R (1981) Promotive effects of ethinyl estradiol in hepatocarcinogenesis initiated by diethylnitrosamine in male rats. Gann, 72: 339
2 Sumi C (1980) Synergism of diethylstilbesterol and other carcinogensin concurrent development of hepatic, mammary and pituitary tumors in castrated male rats. ] Nat! Cancer Inst 65: 169 3 Ding Lian (1982) The promotive effect of diethylstilbesterol on liver cell carcinogenesis initiated by diethylnitrosamine in rat. Chinese] Path 11: 272 (in Chinese) 4 Institute of Laboratory Animal Resources (1980) Histologic ty~ing of liver tumors of the rat. J Nat! Cancer Inst 64: 179 Kalengayi MMR (1975) Histochemistry of gamma-glutamyl transferase in rat liver during aflatoxin B1 induced carcinogenesis. ] Nat! Cancer Inst 55: 579 6 Mitzutani A (1968) Cytochemical demonstration of ornithine carbamyl transferase activity in liver mitochondria of rat and mouse. ] Histochem Cytochem 16: 172 7 Chen ]inyan (1982) Histochemical demonstration of ornithine carbamyl transferase. Chinese ] Path 11: 76 (in Chinese) 8 Reuber MD (1962) Effectof progesterone and diethylstilbesterol on hepatic carcinogenesis and cirrhosis in A X C rats fed N2-fluorenyldiacetamide. J Nat! Cancer Inst 29: 933 9 Li SA (1981) Changes in estrogen receptor levels during DES-induced hepatocarcinogenesis in the Syrian Hamster fed anaPththoflavone. ] Steroid Biochem 15: 387 o Ding Lian (1982) Carcinogenic process and histochemical changes of ornithine carbamyl transferase in rat liver after diethylnitrosamine. Chinese] Path 11: 91; (in Chinese).
Received May 4, 1983 . Accepted August 29, 1983 Ding Lian, Department of Pathology, Institute of BasicMedical Sciences, ChineseAcademyof Medical Sciences, 5 Dong Dan SanTiao, Beijing, PRC
A Histological Study of the Promotive Effect of Diethylstilbestrerol on Diethylnitrosamine Initiated Carcinogenesis of Liver in Rat Ding Lian, Zou Daoyun, Chen Jinyan, Wang Juantan, Wang Yuanbu, Pan Shaoru and Wand Shuhua Department of Pathology, Institute of BasicMedicalSciences, Chinese Academy of Medical Sciences, Beijing, PR China
SUMMARY
A single dose (80 mg/kg) of diethylnitrosamine (DEN) wasgiven orally to castrated female Wistar rats. One week after that one half of the animals were treated with diethylstilbestrol(DES) J mg/kg/once a week subcutaneously. The other half of the animals received no any hormone or hormone derivatives. The change of the liver cells in animals treated with DEN alone failed to progress beyond the stage of hepatocellular alterations in foci or neoplastic nodules ioithm 8 months, while most of those antmals which receiued DES treatment after DEN initiation developedhepatocellular carcinomas after 6 months. This result denotes that the DES exerts a definite promotive effect on DEN initiated liver cell carcinogenesis.
The synthetic estrogens such as ethinyl estradiol and DES have been reported to provide a cocarcinogenic or promotive effect on liver tumorigenesis in some species of animals. Cameron et a1. demon strated that the ethinyl estradiol was a promoter of precancerous liver lesions in male Fischer 344 rats'. Sumi et al. in their experiments showed that the DES played a synergistic effect with Nbutyl-N-nitrosourea or fast neutron (s) irradiation on hepatoma development in castrated male WF rats", We had preliminarily reported that DES provided a promotive effect on liver carcinogenesis in castrated female Wistar rats being formerly initiated by DEN 3• In this experiment the histological changes regarding to the promotive effect of DES in liver carcinogenesis were further studied.
Materials and Methods Seventy two female Wistar rats, 120-150 gms in weight, recieved a single dose of DEN orally (80 mg/kg) one week after castration. Ten rats were sacrificed 3 and 7 days after DEN © 1984 by Gustav FischerVerlag,Stuttgart
administration respectively (5 rats for each) for studying the toxic effect of DEN and the repairing process taking place in the livers. One week after DEN administration, the rats were divided into 2 groups at random. In the first group (DD group) DES was injected subcutaneously in all 31 rats, 3 rng/kg/once a week. In
the second group (D group), 31 rats were used as controls . By the end of the 2nd week as well as the first, 2nd, 4th, 6th and 8th months after DEN, 5 or 7 rats of each group were sacrificed after a fast of 15-17 hours (over night) respectively. Liver tissue blocks were taken from each rat for the following examinations.
1. Routine histopathological study: Tissue blocks, 3 mm in thickness, taken from each lobe of the liver were fixed in 10% formalin for 24 hours. Paraffin sections were stained with hematoxylin and eosin. The histological classification of the hepatic lesions was described in accordance with the nomenclature suggested by The Institute of Laboratory Animal Resources generally used for typing of precancerous and cancerous lesions experimentally induced in rat's liver. 2. Glycogen stain: Tissue blocks taken from left lobe of the livers were fixed and dehydrated in absolute alcohol. The paraffin sections were stained with Best's carmine. 0344-0338/84/0178-0339$3.50/0
340 . Ding Lian, Zou Daoyun, Chen jinyan, Wang juantan, Wang Yuanbu, Pan Shaoru and Wand Shuhua
3. Histochemical demonstration of gamma-glutamyl transferase (GGT) in tissue blocks: Tissue blocks of left and left-middle lobes of liver were taken 4, 6 and 8 months after DEN adminis-
tration. Blocks of 2 mm in thickness, after being rinsed with normal saline (N. S.) and immersed in 0.1 M Tris-HCl buffered saline (pH 7.4) for 5 minutes were incubated in GGT substrate for 1 hour in room temperature. The blocks were rinsed again with N.S.; immersed in 0.1 M copper sulfate solution for 5-10 minutes; then washed with N. S. and were examined under stereomicroscope.
Substate for GGT contained': Garnma-Lglutamyl-c-naphthylamine 5 mg dissolved in 4 ml N.S. Fast blue B 5 mg dissolved in 1 ml N. S. Glycylglycine 5 mg dissolved in 1 ml N. S. 0.1 M Tris-HCl buffered saline, ph 7.4 4 ml. Mixed and filtered immediately before use
4. Histochemical demonstration of GGT and ornithine carbamyltransferase (OCTt· 7: Cryostat sections (7.5-10 11 in thickness) of left middle lobe of liver were fixed in -79 °C. acetone over night. Procedure for GGT was similar as those for tissue block staining. Procedure of histochemical demonstration of OCT: Sections, after fixation, were rinsed with 0.05 M Tris-maleate buffer solution (pH 7.3), and incubated in OCT substrate for 30-35 minutes in room temperature. Substrate for OCT contained: Carbamyl phosphate Li-salt L-ornithine
5 mg 5 mg
0.05 M Tris-maleate buffer solution, pH 7.3 4.5 ml Sucrose 0.8 g Distilled water 5 ml After well mixed 0.5 ml of 2% lead nitrate was added drop by drop under vigerous stirring and then filtered. Substrate must be freshly prepared before use. After that, sections were rinsed with distilled water and immersed in 1% ammonium sulphite for 1 minute. Rinsed the sections with distilled water and stained with 0.5% methyl green if desired and mounted either with glycerin-jelly or dehydrated with alcohol and then mounted with Canada Balsam after xylol for clearing.
Results Three days after DEN administration, the livers slightly swelled to about 3.2% of the body weight (Table 1, normal values: 2.5-3.0%). There was moderate congestion being dark red in colour, and over the cutting surfaces the outline of lobules could still be identified. Microscopically, central and bridging necrosis of liver cells were obtained in nearly all of the lobules. Liver cells over the peripheral zone of the lobules were moderately hyperplastic with mild basophilic cytoplasm, enlarged nuclei, prominent nucleoli and scattered mitotic figures. Mild lymphocytic infiltration presented over the portal spaces but small biliary ductules intacted. OCT activity disappeared in those necrotic liver cells (Fig. 1).
liver weight ) Table 1. Averagecomparative weight of livers ( x 100% in each group (the numbers of animals are quoted in body weight parentheses) Durations after DEN DO group ± S.D. D group ± S.D.
3 days
3.2 (5)
± 0.32
VI M
7days
3.8 (5)
± 1.12
3.8 (5) 3.5 (5)
1M
± 0.51 ± 0.25
4.8 (5) 3.0 (5)
4M
2M
± 1.02 ± 0.19
3.8 (7) 2.7 (5)
± 0.46 ± 0.37
3.2 (5) 2.5 (5)
8M
6M
± 0.13 ± 0.13
4.4 (5) 2.3 (5)
± 0.88 ± 0.27
4.6 (4) 2.3 (6)
± 1.98 ± 0.29
Fig. 1. 3 days after DEN, OCT activity vanished in central necrotic areas, OCT, x 100.
Diethylnitrosamine Initiated Carcinogenesis of Liver in Rat . 341
be easily distinguished from the GGT positive nodules or masses mentioned above. Microscopically, the cytoplasm of the periportal liver cells in 4 months DD group animals were moderately basophilic and their nuclei were deeply stained. Scattered mitotic figures (1-2/HP) might be seen in the midzonal regions as well as large- or multi-nucleated liver cells presented in the central portions of the lobules. Foci and areas of cellular alterations similar as those seen in animals of single DEN group were also obtained in the sections (Fig. 2). Neoplastic nodules began to develop in 2 of the 5 DD group animals 4 months after DEN and they grew up to form varying sized nodules in all of the DD animals 6 months after DEN administration (Fig. 3). In a few nodules the basophilic liver cells looked invading into the surrounding tissue which was considered to be a criteria of undergoing malignant change. Trabecular hepatocellular carcinomas were found in 4 out of 5 rats in DD group after 6 months of DEN intake and in 3 out of 4 animals after 8 months of DEN. These cancer cells arranged in cords, acini or masses and their cytoplasm revealed basophilic, nuclei deeply stained and nucleoli increased or enlarged. The mitotic figures were numerous in the cancer nests (4-5/HP) (Fig. 4). The borders of these cancer masses were ill-defined and Best's carmine stain revealed no apparent glycogen storage in most of the cancer cells. GGT reaction of these cancer cells showed strong positive (Fig. 5) while OCT activity was markedly decreased or vanished (Fig. 6). Cirrhosis of liver obtained in one DD rat of the 8th month group and OCT activity of the liver cells in cirrhotic nodules was positive.
Seven days after DEN administration, livers became moderately enlarged making up 3.8% of the body weight (Table 1). Microscopically, most of the central necrosis disappeared, only occasional necrotic or eosinophilic degenerated liver cells found surrounding the central veins. Numerous binucleated liver cells and scattered mitotic figures still presented in the peripheral and midzonal regions of the lobules. OCT activities repaired to normal range in most of the lobules. Within 2 months of DEN administration, livers of animals in both groups slightly or moderately enlarged (Table 1). Microscopically, all these livers revealed essentially normal. The OCT activities also showed normal. After 4 months of DEN administration, livers of those rats recieved DEN alone almostly recovered to normal in appearence grossly and GGT reactions showed no positive signs or only got occasional red spots in the gross specimens (Table 2). Microscopically, both liver cells and lobules were well preserved except more or less foci and areas of hepatocellular alterations seen (Table 2). Most of these foci consisted of clear and acidophilic liver cells while those foci composed of basophilic cells did not exceed 10% totally. Best's carmine stain showed marked storage of glycogen in the clear and acidophilic liver cells but OCT activities decreased or disappeared. Livers of rats in DD group were larger than the normal ones and progresively increased their weight until up to nearly twice the weight of the normals after 6 months of DEN administration (Table 1). Macroscopically, these livers showed dark red in colour and granular in appearence with dullrounded edges. The cutting surfaces were markedly bulging up with scattered gray-yellowish granules, nodules or masses. GGT reaction gave these granules, nodules or masses reddish in colour which would be easily recognized and counted under stereomicroscope. The number of the GGT positive granules and nodules of DD group animals were apparently more than those of the D group animals (Table 2). In some cases of DD group, the periportal liver tissue might also show a red colour in GGT reaction giving a nutmeg in appearence. These streak mottled areas could
Discussion DES have been used as an estrogen substitute clinically. Experimentally, it is known to have the abilities in inducing various tumors such as tumors of uterine cervix and kidneys in rodents. Formerly, Reuber et al.8 reported that DES provided a protective effect on N-2-fluorenyldiacetamide induced cirrhosis of liver and reduced the
Table 2. Hepatic lesions of the rats Months after DEN Groups Number of animals
2 DD 7
D 5
GGT (+) foci in gross specimen avo no.lcm 2 ± S.D. Foci of cellular alterations avo no.zcrrr' 0.8 ± S.D. 10.9 % of basophilic cell foci ± S.D.
0.7 0
4 DD 5
6 DD 5
D 5
8 DD 4
D 5
D 6
1.0 ±0.6
0
4.6 ±2.8
0.9 ±0.8
3.3 ± 2.2
0.9 ±0.5
1.0 ±0.4 25.5 ± 10.8
2.1 ±1.3 7.2 ±7.0
6.2 ±2.8 26.9 ± 11.2
2.3 ±1.4 4.0
4.7 ± 1.8 29.5 ± 11.4
3.5 ±2.3 7.8 ±6.7
Neoplastic nodule"
0
0
2
0
5
0
4
1
Liver cell cancer" *
0
0
0
0
4
0
3
0
* Number of rats developed neoplastic nodules ** Number of rats developed liver cell carcinomas
342 . Ding Lian, Zou Daoyun, Chen jin yan, Wang j uantan , Wang Yuanbu, Pan Shaoru and Wand Shuhua
Fig. 2. DD group, 6 month s after DEN, neoplastic nodule composed of vacuolated clear liver cells, HE, X 100.
Fig. 3. DD group , 6 months after DEN, neoplastic nodule composed 'o f basophilic liver cells, HE, X 100.
Fig. 4. DD group, 8 months after DEN, hepatocellular carcinoma and cirrhosis of liver, HE, X 100.
Diethylnitrosamine Initiated Carcinogenesis of Liver in Rat . 343
Fig. 5. DD group, 6 months after DEN, Liver cell cancer shows GGT positive reaction. GGT, X 100.
Fig. 6. DD group, 8 months after DEN, OCT activity markedly decreased in liver cell cancer. OGT, X 100. incidence of hepatoma in A X C male rats. Recently, works denoted that DES was a cocarcinogen of liver in castrated male rats and DES alone might induce a definite hepatocarcinogenicity in those rats even with only a low incidence of liver cells tumor', In this experiment, the livers after a single dose (80 mg/kg) of DEN failed to progress beyond lesions of altered cell foci and occasional neoplastic nodule within 8 months. If the animals were treated with DES persistently for 4 months after DEN initiation there would be marked hyperplasia of periportal liver cells and numerous basophilic altered cell foci present in livers and neoplastic nodules began to develop in these animals. If DES giving was lasted for more than 6 months, not only all the animals developed neoplastic nodules but also hepatocellular cancers formed in most of these animals. The results denote that DES exerts a promotive effect on DEN initiated liver carcinogenesis in castrated female Wistar rats.
DES is a synthetic analogue of estrogen. Li et al." recently reported that a high incidence (70%) of hepatoma in castrated male Syrian Hamsters were obtained after treatment with DES plus a-naphthoflavone (a-NF), an inhibitor of microsomal mixed function oxidase. Although the estrogen receptors of these liver cells elevated up to about 5-fold, anyhow, no difference was obtained in estrogen receptor levels in comparing with those found in other species of animals. Therefore the authors considered that the induction of hepatoma by DES plus a-NF in these hamsters was possibly due to a non-hormonal mechanism. Sumi et al.2 considered that the DES might be a weak carcinogen or might exert as a promotor indirectly by its induction of hypersecretion of pituitary hormone such as the secreta of mammosomatotropic pituitary tumor which have been shown to be a promoter of chemical carcinogen induced hepatocarcinogenesis. Whether the promotive role of DES on hepatocarcinogenesis is due to its chemical
344 . Ding Lian, Zou Daoyun, Chen ]inyan, Wang ]uantan, Wang Yuanbu, Pan Shaoru and Wand Shuhua character (as a weak carcinogen) or due to its hormonal effect (enhancement of protein synthesis or acceleration of hyperplasia of liver cells) or owing to both still remains to be studied. GGT reaction in tissue blocks shows some altered liver cell foci, neoplastic hepatocellular nodules and cancer masses in red colour and makes these lesions easily be recognized by naked eye or under stereomicroscope particularly if these are only a few small nodules presented on the cutting surfaces. The OCT activities of immature or embryonal liver cells are very weak or even negative. The precancerous and cancerous hepatocytes also reveal lowered or vanished OCT activity. Sometimes the degenerated or necrotic liver cells may also give a weak or vanished OCT reaction, but the positive GGT reaction only presents in proliferating liver cells. Therefore it seems better to evaluate the OCT negative lesions accompanying with the result of GGT reaction for making a more definite decision of proliferating immature liver cells lesions. References 1 Cameron R (1981) Promotive effects of ethinyl estradiol in hepatocarcinogenesis initiated by diethylnitrosamine in male rats. Gann, 72: 339
2 Sumi C (1980) Synergism of diethylstilbesterol and other carcinogensin concurrent development of hepatic, mammary and pituitary tumors in castrated male rats. ] Nat! Cancer Inst 65: 169 3 Ding Lian (1982) The promotive effect of diethylstilbesterol on liver cell carcinogenesis initiated by diethylnitrosamine in rat. Chinese] Path 11: 272 (in Chinese) 4 Institute of Laboratory Animal Resources (1980) Histologic ty~ing of liver tumors of the rat. J Nat! Cancer Inst 64: 179 Kalengayi MMR (1975) Histochemistry of gamma-glutamyl transferase in rat liver during aflatoxin B1 induced carcinogenesis. ] Nat! Cancer Inst 55: 579 6 Mitzutani A (1968) Cytochemical demonstration of ornithine carbamyl transferase activity in liver mitochondria of rat and mouse. ] Histochem Cytochem 16: 172 7 Chen ]inyan (1982) Histochemical demonstration of ornithine carbamyl transferase. Chinese ] Path 11: 76 (in Chinese) 8 Reuber MD (1962) Effectof progesterone and diethylstilbesterol on hepatic carcinogenesis and cirrhosis in A X C rats fed N2-fluorenyldiacetamide. J Nat! Cancer Inst 29: 933 9 Li SA (1981) Changes in estrogen receptor levels during DES-induced hepatocarcinogenesis in the Syrian Hamster fed anaPththoflavone. ] Steroid Biochem 15: 387 o Ding Lian (1982) Carcinogenic process and histochemical changes of ornithine carbamyl transferase in rat liver after diethylnitrosamine. Chinese] Path 11: 91; (in Chinese).
Received May 4, 1983 . Accepted August 29, 1983 Ding Lian, Department of Pathology, Institute of BasicMedical Sciences, ChineseAcademyof Medical Sciences, 5 Dong Dan SanTiao, Beijing, PRC