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Importance of the mouse liver tumor in carcinogenesis bioassay studies using benzidine dihydrochloride as a model
Toxicology Letters, 4 (1979) 507-518 o Elsevier/North-Holland Biomedical Press
507
IMPORTANCE OF THE MOUSE LIVER TUMOR IN CARCINOGENESIS BIOASSAY STUDIES USING BENZIDINE DIHYDROCHLORIDE AS A MODEL
CHARLES H. FRITH*,***, KARL P. BAETCKE**, GEORGE SCHIFSFERSTEIN***
C.J. NELSON***
and
*Department of Pathology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72201; Pathology Services Project, National Center for Toxicological Research, Jefferson, AR 72079; **3715 Dora1 Drive, Little Rock, AR, and ***Department of Health, Education and Welfare, Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079 (U.S.A.) (Received August 16th, 1979) (Accepted August 19th, 1979)
SUMMARY
Benzidine dihydrochloride induced hepatocellular adenomas in male and female mice which appeared to progress to hepatocellular carcinomas as either the length of administration and/or the dose level of benzidine increased. The differentiation of hepatocellular carcinomas also correlated inversely with dose, and poorly differentiated hepatocellular carcinomas metastasized more frequently. These findings support the view that the mouse liver tumor is predictive of carcinogenicity in bioassay studies.
INTRODUCTION
Neoplasms and other histopathological lesions are frequently seen in the livers of mice following the administration of carcinogens [ 5,9, 121. Evaluation of the biological significance of liver lesions is important in determining whether the mouse can be utilized in assessing the potential carcinogenicity of suspect compounds in bioassay studies. In some studies involving hepatocarcinogens in mice the lesions have been designated hepatomas or liver neoplasms without adequate discussion of their significance. The main objective of the present study was to assess the biological significance of liver tumors in mice by correlating (1) benign liver neoplasms; (2) malignant liver neoplasms; (3) the degree of differentiation of malignant neoplasms; and (4) pulmonary metastases, with reference to dose level and length of administration of benzidine dihydrochloride. *Reprint request to Dr. Frith at NCTR.
508 TABLE
I
EXPERIMENTAL
DESIGN
Dose level of benzidine (ppm)
Length of administration (Weeks) 40
0 30 60 120 200 400 Total
-
60
80
Number of animals 48 48 48 96 72 48 12 48 48 48 48 48 48 48 24 24 24 24
144 216 168 144 120 72
336
864a
288
240
a2 crosses and 2 sexes = 2 X 2 X 864 = 3456 MATERIALS
Total
-
animals.
AND METHODS
3456 F1 (C57BL/GJfC3Hf/Nctr females X BALB/cStCrlfC3Hf/Nctr males) and F2 (F, females X F, males) mice (Table I) were used. Weanling animals were housed, 4 per cage, in a room maintained at 22-24°C. Food and water were provided ad libitum. The animals were fed Purina 501OC meal and dosed with water containing 0,30,60,120,200 and 400 ppm of benzidine dihydrochloride (Allied Chemical Co., Buffalo, NY). No deviation from our standard benzidine dehydrochloride was detected, and it was considered to be essentially 100% pure. Groups of mice were killed at 40, 60 and 80 weeks for pathologicalevaluation. At death, each animal was given a carcass identification (CID) number. Detailed necropsies were performed, and gross and microscopic findings were collected on ca. 45 tissues or organs of each animal as described by Frith et al. [ 31. Sections of grossly visible lesions and of the median, left and right lateral lobes of the liver were also collected. After fixation in Bouin’s solution for 18-24 h the tissues were routinely trimmed, placed in plastic cassettes for processing on an automatic tissue processor on a 4-h cycle, and embedded in paraffin blocks. Routine paraffin sections were cut at 5 pm and stained on an automatic stainer with hematoxylin and eosin (H & E) as described by Frith et al. [3] . RESULTS
This study revealed a variety of histopathological lesions including liver neoplasms, which will be reported in detail separately and which were similar to those previously reported [2] . An animal that died prior to its scheduled killing was included in the scheduled period closest to its death. Since no significant differences were observed between the F, and FZ males and those in the F, and Fz females have been combined. Hepatocellular neoplasms were classified as either adenomas or carcinomas
Fig. 1. Photomicrograph of a hepatoceIlular adenoma (top) which is causing compression of liver parenchyma (bottom). H&E x 350. Fig. 2, PhotQrnj~~~~ap~ of trabecular pattern commonly associated with hepatoceBul% carcinoma. H&E x 208.
based upon the classification system currently in use at NCTR [4]. The hepatocellular adenomas were usually small (< 5 mm) and existed as distinct nodules which compressed adjacent normal hepatic parenehyma (Fig. 1). Histologically they were composed of well-differentiated eelIs which showed little pleomorphism, were uniform in size and sometimes formed regular cords one cell thick. The hepatie curds were lined by sinusoidal. cells, but the sinusoids were not prominent. ~epat~~ellul~ c~ci~~rn~ were disposed on the basis of either a distinct trabecular or adenoid pattern (Fig. 2) or of cytological features, characteristic of malignancy. The liver cell plates were more than one cell layer thick, irregular and composed of well to poorly differentiated hepatocytes. When hepato~e~~u~~adenomas and carcinomas occurred in the same liver, they were included only in the malignant tabulations. The ratio of he~~tocellul~ c~~inorn~ to he~~t~cellul~ ad~arn~ increased both with length of administration (Chart 1) and dose level (Chart 2f of the carcinogen in females of both strains and with time, but not with dose in the males of both strains (Charts 3 and 4). The total number of liver neoplasms was higher in the females but the ratio of malignant liver neoplasms : adenomas was not s~~ifi~antly different between females and males. At 0 ppm the ratio of beady to rn~i~a~t hepatic t~rno~ was 67:33 and 50:50 for males and females, respectively; at 400 ppm it was 25:75 and 15:85 for males and females, respectively. Hepato~ellu~~ carcinomas were separated into well, moderately welf and poorly differentiated &asses. If more than one h~pato~e~u~~ carcinoma w&s
Chart. 1. Ratio of hepatocellular adenomas to hepatocellular ministration of carcinogen (F, and F, females).
Chart 2. Ratio of hepatocellular carcinogen (F, and F, females).
adenomas
to hepatocellular
carcinomas
carcinomas
with length
of ad-
with dose level of
511
Chart 3. Ratio of hepatocellular adenomas to hepatocellular administration of carcinogen (F, and ‘F, males).
0
IQ
Chart 4. Ratio of hepatocellular carcinogen (F, and F, males).
60
120
adenomas to hepatocellular
carcinomas with length of
200
400
carcinomas to dose level of
512
Fig. 3. Photomicrograph of a well differentiated hepatocellular carcinoma demonstrating a trabecular pattern and a relatively uniform cell population. H&E X 350. Fig. 4. Photomicrograph of a moderately well differentiated hepatocellular carcinoma composed of larger less uniform hepatocytes. H&E X 350.
present in a single liver, only the least differentiated was recorded. The well differentiated hepatocellular carcinomas were composed of uniform cells with a fair amount of cytoplasm. A distinct trabecular pattern was often evident (Fig. 3). The moderately well differentiated hepatocellular carcinomas were usually composed of larger cells which varied more in size and shape (Fig. 4). The poorly differentiated tumors were composed of either small immature hepatocytes (Fig. 5) or extremely large anaplastic cells. Well differentiated neoplasms did not differ in incidence significantly at the different periods of investigation, but poorly differentiated neoplasms were seen only at 60 and 80 weeks (Charts 5 and 6). The incidence of moderately well and poorly differentiated hepatocellular carcinomas increased significantly with an increase in dose level of the carcinogen in the females, but not in the males (Charts 7 and 8). The degree of differentiation of the malignant liver neoplasms also correlated with the incidence of pulmonary metastases (Fig. 6). The incidence of metastases for well, moderately well, and poorly differentiated hepatocellular carcinomas was 5,lO and 14% respectively (Chart 9). The incidence of pulmonary metastases also increased with the length of administration of the carcinogen. No metastases were found at 40 weeks; of the 34 liver tumors which metastasized, 12 of 239 (5.0%) hepatocellular carcinomas metastasized at 60 weeks and 22 of 186 (11.8%) metastasized at 80 weeks (Chart 10).
Fig. 5. Photomicrograph of a poorly differentiated small immature hepatocytes. H&E X 350. Fig. 6. Photomicrograph
hepatocellular
of pulmonary metastasis of hepatocellular
Chart 5. Frequency of degree of differentiation of hepatocellular of administration of carcinogen (F, and F, males).
carcinoma composed of
carcinoma.
H&E
x
350.
carcinomas with length
514
40
60
Chart 6. Frequency of degree of differentiation of hepatocellular administration of carcinogen (F, and F, females).
30
60
120
Chart 7. Frequency of degree of differentiation level of carcinogen (F, and F, females).
200
of hepatocellular
carcinoma with length of
400
carcinomas with dose
515
100
00
80
IQ
60
50
5 y = 1
IQ
z e=
30
20
IQ
0
Chart 8. Frequency of degree of differentiation level of carcinogen (F, and F, males).
of hepatocellular
carcinomas with dose
25
20
Well
‘Actual
Number
of Hepatacellular
Moderately
Cawnomar
IS represented
well
by number
in battom
of each
bar
Chart 9. Frequency of degree of differentiation of hepatocellular of pulmonary metastasis (F, and F, males and females).
carcinomas with incidence
516
40
Chart 10. Frequency of incidence of pulmonary metastasis with length of administration of carcinogen (F, and F, males and females).
No association between incidence of metastases and dose level of the carcinogen was detected. There was also no difference in the incidence of metastases between sexes. The total incidence of pulmonary metastases was 6.9% and 7.9% for the males and females, respectively, DISCUSSION
This study confirms the carcinogenicity of benzidine in mice reported by earlier investigators [ 1, 2, 7, lo] . Two crosses were utilized to test the hypothesis that the Fz cross would be more representative of the human population because of its genetic heterogeneity compared to the genetic homogeneity of the F, cross. The two crosses tended to respond similarly, and differences between the sexes were greater than differences between the crosses. The ratio of malignant to benign liver neoplasms increased in both sexes of both crosses with the length of administration of the carcinogen and with the dose level in the females of both strains. That the ratio of malignant to benign liver tumors increased with both dose level and length of administration is regarded as evidence that malignant liver tumors in the mouse develop as a progression from benign to malignant. Similar findings have been previously reported using less detailed methods of evaluation [4,13] . The degree of differentiation correlated well in the F, and Fz females, but
517
poorly in the males, with the dose level of carcinogen. The degree of differentiation also correlated with the incidence of metastases. Although the total number of moderately and poorly differentiated hepatocellular carcinomas was smaller than that of the well differentiated, the incidence of metastases was greater. This is in agreement with the biological behavior of malignant neoplasms in other species and adds support for use of the liver tumor in the mouse for predicting carcinogenicity in bioassay studies. The incidence of metastases has generally been considered very low for mouse liver tumors and is used as an argument against the utilization of the mouse liver tumor as indicative of carcinogenicity in bioassay studies. Recent studies have however indicated that a thorough examination of lung sections reveals a much higher incidence than previously suspected 16,111. This has been confirmed in the present study. The incidence averaged ca. 8.0% in males and females of both crosses, based upon the examination of only a single section of the lungs. Additional sections would probably have resulted in a higher incidence of metastases. A direct correlation did not exist between dose level and incidence of pulmonary metastases, but the incidence of pulmonary metastases correlated with the degree of differentiation and the length of administration of the carcinogen. Our results indicate that liver neoplasms in mice appear early as benign hepatocellular adenomas which later may progress to morphologically malignant hepatocellular carcinomas. The hepatocellular carcinomas may become less differentiated with time and dose and metastasize. Liver neoplasms in the mouse appear to behave in a manner similar to neoplasms in other species. Our study supports the view that the mouse liver tumor is predictive of carcinogenicity in bioassay studies [8] . REFERENCES 1 G.M. Bonser, D.V. Clayson and J.W. Jull, The induction of tumors of the subcutaneous tissue, liver and intestine in the mouse by certain dyestuffs and their intermediates, Br. J. Cancer, 10 (1956) 653-667. 2 C.H. Frith and K. Dooley, Hepatic cytologic and neoplastic changes in mice given benzidine dihydrochloride, J. Natl. Cancer Inst., 56 (1976) 679-682. 3 C.H. Frith, B. Highman and A.J. Konvicka, Advances in automation for experimental pathology, Lab. Anim. Sci., 26 (1976) 171-185. 4 C.H. Frith and J.M. Ward, A morphologic classification of proliferative and neoplastic hepatic lesions in mice, J. Environ. Tox. Pathol. In Press. 5 J.R.M. Innes, B.M. Ulland, M.G. Valerio, L. Petrucelli, L. Fishbein, E.R. Hart, A.J. Pallotta, R.R. Bates, H.L. Falk, J.J. Gart, M. Klein, I. Mitchell and J. Peters, Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note, J. Natl. Cancer Inst., 42 (1969) 1101-1114. 6 A.P. Kyriazis, M. Koda and S.D. Wesselinovitch, Metastatic rate of liver tumors induced by diethylnitrosamine in mice, Cancer Res., 34 (1974) 2881-2886. 7 O.G. Prokofjeva, Induction of hepatic tumors in mice by benzidine, Vopr. Onkol., 17 (1971) 61-64. 8 L. Tomatis, C. Partensky and R. Montesano, The predictive value of mouse liver tumour induction in carcinogenicity testing -a literature survey, Int. J. Cancer, 12 (1973) l-20.
518 9 L. Tomatis, V. Turosov, R.T. Charles, M. Boiocchi and E. Gati, Liver tumors in CF-1 mice exposed for limited periods to technical DDT, Z. Krebsforsch., 82 (1972) 25-35. 10 S.D. Vesselinovitch, K.V.N. Rao and N. Mihailovich, Factors modulating benzidine carcinogenicity bioassay, Canad. Res., 35 (1975) 2814-2819. 11 S.D. Vesselinovitch, N. Mihailovich and K.V.N. Rao, Morphology and metastatic nature of induced hepatic nodular lesions in C57BLxC3HF, mice, Canad. Res., 38 (1978) 2003-2010. 12 A.I.T. Walker, E. Thorpe and D.E. Stevenson, The toxicology of Dieldrin (HEOD) I. Long-term oral toxicity studies in mice, Food Cosmet. Toxicol., 11 (1972) 415-432. 13 J.M. Ward and G. Vlahakis, Evaluation of hepatocellular neoplasms in mice, J. Natl. Cancer Inst., 61 (1978) 807-809.
507
IMPORTANCE OF THE MOUSE LIVER TUMOR IN CARCINOGENESIS BIOASSAY STUDIES USING BENZIDINE DIHYDROCHLORIDE AS A MODEL
CHARLES H. FRITH*,***, KARL P. BAETCKE**, GEORGE SCHIFSFERSTEIN***
C.J. NELSON***
and
*Department of Pathology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR 72201; Pathology Services Project, National Center for Toxicological Research, Jefferson, AR 72079; **3715 Dora1 Drive, Little Rock, AR, and ***Department of Health, Education and Welfare, Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079 (U.S.A.) (Received August 16th, 1979) (Accepted August 19th, 1979)
SUMMARY
Benzidine dihydrochloride induced hepatocellular adenomas in male and female mice which appeared to progress to hepatocellular carcinomas as either the length of administration and/or the dose level of benzidine increased. The differentiation of hepatocellular carcinomas also correlated inversely with dose, and poorly differentiated hepatocellular carcinomas metastasized more frequently. These findings support the view that the mouse liver tumor is predictive of carcinogenicity in bioassay studies.
INTRODUCTION
Neoplasms and other histopathological lesions are frequently seen in the livers of mice following the administration of carcinogens [ 5,9, 121. Evaluation of the biological significance of liver lesions is important in determining whether the mouse can be utilized in assessing the potential carcinogenicity of suspect compounds in bioassay studies. In some studies involving hepatocarcinogens in mice the lesions have been designated hepatomas or liver neoplasms without adequate discussion of their significance. The main objective of the present study was to assess the biological significance of liver tumors in mice by correlating (1) benign liver neoplasms; (2) malignant liver neoplasms; (3) the degree of differentiation of malignant neoplasms; and (4) pulmonary metastases, with reference to dose level and length of administration of benzidine dihydrochloride. *Reprint request to Dr. Frith at NCTR.
508 TABLE
I
EXPERIMENTAL
DESIGN
Dose level of benzidine (ppm)
Length of administration (Weeks) 40
0 30 60 120 200 400 Total
-
60
80
Number of animals 48 48 48 96 72 48 12 48 48 48 48 48 48 48 24 24 24 24
144 216 168 144 120 72
336
864a
288
240
a2 crosses and 2 sexes = 2 X 2 X 864 = 3456 MATERIALS
Total
-
animals.
AND METHODS
3456 F1 (C57BL/GJfC3Hf/Nctr females X BALB/cStCrlfC3Hf/Nctr males) and F2 (F, females X F, males) mice (Table I) were used. Weanling animals were housed, 4 per cage, in a room maintained at 22-24°C. Food and water were provided ad libitum. The animals were fed Purina 501OC meal and dosed with water containing 0,30,60,120,200 and 400 ppm of benzidine dihydrochloride (Allied Chemical Co., Buffalo, NY). No deviation from our standard benzidine dehydrochloride was detected, and it was considered to be essentially 100% pure. Groups of mice were killed at 40, 60 and 80 weeks for pathologicalevaluation. At death, each animal was given a carcass identification (CID) number. Detailed necropsies were performed, and gross and microscopic findings were collected on ca. 45 tissues or organs of each animal as described by Frith et al. [ 31. Sections of grossly visible lesions and of the median, left and right lateral lobes of the liver were also collected. After fixation in Bouin’s solution for 18-24 h the tissues were routinely trimmed, placed in plastic cassettes for processing on an automatic tissue processor on a 4-h cycle, and embedded in paraffin blocks. Routine paraffin sections were cut at 5 pm and stained on an automatic stainer with hematoxylin and eosin (H & E) as described by Frith et al. [3] . RESULTS
This study revealed a variety of histopathological lesions including liver neoplasms, which will be reported in detail separately and which were similar to those previously reported [2] . An animal that died prior to its scheduled killing was included in the scheduled period closest to its death. Since no significant differences were observed between the F, and FZ males and those in the F, and Fz females have been combined. Hepatocellular neoplasms were classified as either adenomas or carcinomas
Fig. 1. Photomicrograph of a hepatoceIlular adenoma (top) which is causing compression of liver parenchyma (bottom). H&E x 350. Fig. 2, PhotQrnj~~~~ap~ of trabecular pattern commonly associated with hepatoceBul% carcinoma. H&E x 208.
based upon the classification system currently in use at NCTR [4]. The hepatocellular adenomas were usually small (< 5 mm) and existed as distinct nodules which compressed adjacent normal hepatic parenehyma (Fig. 1). Histologically they were composed of well-differentiated eelIs which showed little pleomorphism, were uniform in size and sometimes formed regular cords one cell thick. The hepatie curds were lined by sinusoidal. cells, but the sinusoids were not prominent. ~epat~~ellul~ c~ci~~rn~ were disposed on the basis of either a distinct trabecular or adenoid pattern (Fig. 2) or of cytological features, characteristic of malignancy. The liver cell plates were more than one cell layer thick, irregular and composed of well to poorly differentiated hepatocytes. When hepato~e~~u~~adenomas and carcinomas occurred in the same liver, they were included only in the malignant tabulations. The ratio of he~~tocellul~ c~~inorn~ to he~~t~cellul~ ad~arn~ increased both with length of administration (Chart 1) and dose level (Chart 2f of the carcinogen in females of both strains and with time, but not with dose in the males of both strains (Charts 3 and 4). The total number of liver neoplasms was higher in the females but the ratio of malignant liver neoplasms : adenomas was not s~~ifi~antly different between females and males. At 0 ppm the ratio of beady to rn~i~a~t hepatic t~rno~ was 67:33 and 50:50 for males and females, respectively; at 400 ppm it was 25:75 and 15:85 for males and females, respectively. Hepato~ellu~~ carcinomas were separated into well, moderately welf and poorly differentiated &asses. If more than one h~pato~e~u~~ carcinoma w&s
Chart. 1. Ratio of hepatocellular adenomas to hepatocellular ministration of carcinogen (F, and F, females).
Chart 2. Ratio of hepatocellular carcinogen (F, and F, females).
adenomas
to hepatocellular
carcinomas
carcinomas
with length
of ad-
with dose level of
511
Chart 3. Ratio of hepatocellular adenomas to hepatocellular administration of carcinogen (F, and ‘F, males).
0
IQ
Chart 4. Ratio of hepatocellular carcinogen (F, and F, males).
60
120
adenomas to hepatocellular
carcinomas with length of
200
400
carcinomas to dose level of
512
Fig. 3. Photomicrograph of a well differentiated hepatocellular carcinoma demonstrating a trabecular pattern and a relatively uniform cell population. H&E X 350. Fig. 4. Photomicrograph of a moderately well differentiated hepatocellular carcinoma composed of larger less uniform hepatocytes. H&E X 350.
present in a single liver, only the least differentiated was recorded. The well differentiated hepatocellular carcinomas were composed of uniform cells with a fair amount of cytoplasm. A distinct trabecular pattern was often evident (Fig. 3). The moderately well differentiated hepatocellular carcinomas were usually composed of larger cells which varied more in size and shape (Fig. 4). The poorly differentiated tumors were composed of either small immature hepatocytes (Fig. 5) or extremely large anaplastic cells. Well differentiated neoplasms did not differ in incidence significantly at the different periods of investigation, but poorly differentiated neoplasms were seen only at 60 and 80 weeks (Charts 5 and 6). The incidence of moderately well and poorly differentiated hepatocellular carcinomas increased significantly with an increase in dose level of the carcinogen in the females, but not in the males (Charts 7 and 8). The degree of differentiation of the malignant liver neoplasms also correlated with the incidence of pulmonary metastases (Fig. 6). The incidence of metastases for well, moderately well, and poorly differentiated hepatocellular carcinomas was 5,lO and 14% respectively (Chart 9). The incidence of pulmonary metastases also increased with the length of administration of the carcinogen. No metastases were found at 40 weeks; of the 34 liver tumors which metastasized, 12 of 239 (5.0%) hepatocellular carcinomas metastasized at 60 weeks and 22 of 186 (11.8%) metastasized at 80 weeks (Chart 10).
Fig. 5. Photomicrograph of a poorly differentiated small immature hepatocytes. H&E X 350. Fig. 6. Photomicrograph
hepatocellular
of pulmonary metastasis of hepatocellular
Chart 5. Frequency of degree of differentiation of hepatocellular of administration of carcinogen (F, and F, males).
carcinoma composed of
carcinoma.
H&E
x
350.
carcinomas with length
514
40
60
Chart 6. Frequency of degree of differentiation of hepatocellular administration of carcinogen (F, and F, females).
30
60
120
Chart 7. Frequency of degree of differentiation level of carcinogen (F, and F, females).
200
of hepatocellular
carcinoma with length of
400
carcinomas with dose
515
100
00
80
IQ
60
50
5 y = 1
IQ
z e=
30
20
IQ
0
Chart 8. Frequency of degree of differentiation level of carcinogen (F, and F, males).
of hepatocellular
carcinomas with dose
25
20
Well
‘Actual
Number
of Hepatacellular
Moderately
Cawnomar
IS represented
well
by number
in battom
of each
bar
Chart 9. Frequency of degree of differentiation of hepatocellular of pulmonary metastasis (F, and F, males and females).
carcinomas with incidence
516
40
Chart 10. Frequency of incidence of pulmonary metastasis with length of administration of carcinogen (F, and F, males and females).
No association between incidence of metastases and dose level of the carcinogen was detected. There was also no difference in the incidence of metastases between sexes. The total incidence of pulmonary metastases was 6.9% and 7.9% for the males and females, respectively, DISCUSSION
This study confirms the carcinogenicity of benzidine in mice reported by earlier investigators [ 1, 2, 7, lo] . Two crosses were utilized to test the hypothesis that the Fz cross would be more representative of the human population because of its genetic heterogeneity compared to the genetic homogeneity of the F, cross. The two crosses tended to respond similarly, and differences between the sexes were greater than differences between the crosses. The ratio of malignant to benign liver neoplasms increased in both sexes of both crosses with the length of administration of the carcinogen and with the dose level in the females of both strains. That the ratio of malignant to benign liver tumors increased with both dose level and length of administration is regarded as evidence that malignant liver tumors in the mouse develop as a progression from benign to malignant. Similar findings have been previously reported using less detailed methods of evaluation [4,13] . The degree of differentiation correlated well in the F, and Fz females, but
517
poorly in the males, with the dose level of carcinogen. The degree of differentiation also correlated with the incidence of metastases. Although the total number of moderately and poorly differentiated hepatocellular carcinomas was smaller than that of the well differentiated, the incidence of metastases was greater. This is in agreement with the biological behavior of malignant neoplasms in other species and adds support for use of the liver tumor in the mouse for predicting carcinogenicity in bioassay studies. The incidence of metastases has generally been considered very low for mouse liver tumors and is used as an argument against the utilization of the mouse liver tumor as indicative of carcinogenicity in bioassay studies. Recent studies have however indicated that a thorough examination of lung sections reveals a much higher incidence than previously suspected 16,111. This has been confirmed in the present study. The incidence averaged ca. 8.0% in males and females of both crosses, based upon the examination of only a single section of the lungs. Additional sections would probably have resulted in a higher incidence of metastases. A direct correlation did not exist between dose level and incidence of pulmonary metastases, but the incidence of pulmonary metastases correlated with the degree of differentiation and the length of administration of the carcinogen. Our results indicate that liver neoplasms in mice appear early as benign hepatocellular adenomas which later may progress to morphologically malignant hepatocellular carcinomas. The hepatocellular carcinomas may become less differentiated with time and dose and metastasize. Liver neoplasms in the mouse appear to behave in a manner similar to neoplasms in other species. Our study supports the view that the mouse liver tumor is predictive of carcinogenicity in bioassay studies [8] . REFERENCES 1 G.M. Bonser, D.V. Clayson and J.W. Jull, The induction of tumors of the subcutaneous tissue, liver and intestine in the mouse by certain dyestuffs and their intermediates, Br. J. Cancer, 10 (1956) 653-667. 2 C.H. Frith and K. Dooley, Hepatic cytologic and neoplastic changes in mice given benzidine dihydrochloride, J. Natl. Cancer Inst., 56 (1976) 679-682. 3 C.H. Frith, B. Highman and A.J. Konvicka, Advances in automation for experimental pathology, Lab. Anim. Sci., 26 (1976) 171-185. 4 C.H. Frith and J.M. Ward, A morphologic classification of proliferative and neoplastic hepatic lesions in mice, J. Environ. Tox. Pathol. In Press. 5 J.R.M. Innes, B.M. Ulland, M.G. Valerio, L. Petrucelli, L. Fishbein, E.R. Hart, A.J. Pallotta, R.R. Bates, H.L. Falk, J.J. Gart, M. Klein, I. Mitchell and J. Peters, Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note, J. Natl. Cancer Inst., 42 (1969) 1101-1114. 6 A.P. Kyriazis, M. Koda and S.D. Wesselinovitch, Metastatic rate of liver tumors induced by diethylnitrosamine in mice, Cancer Res., 34 (1974) 2881-2886. 7 O.G. Prokofjeva, Induction of hepatic tumors in mice by benzidine, Vopr. Onkol., 17 (1971) 61-64. 8 L. Tomatis, C. Partensky and R. Montesano, The predictive value of mouse liver tumour induction in carcinogenicity testing -a literature survey, Int. J. Cancer, 12 (1973) l-20.
518 9 L. Tomatis, V. Turosov, R.T. Charles, M. Boiocchi and E. Gati, Liver tumors in CF-1 mice exposed for limited periods to technical DDT, Z. Krebsforsch., 82 (1972) 25-35. 10 S.D. Vesselinovitch, K.V.N. Rao and N. Mihailovich, Factors modulating benzidine carcinogenicity bioassay, Canad. Res., 35 (1975) 2814-2819. 11 S.D. Vesselinovitch, N. Mihailovich and K.V.N. Rao, Morphology and metastatic nature of induced hepatic nodular lesions in C57BLxC3HF, mice, Canad. Res., 38 (1978) 2003-2010. 12 A.I.T. Walker, E. Thorpe and D.E. Stevenson, The toxicology of Dieldrin (HEOD) I. Long-term oral toxicity studies in mice, Food Cosmet. Toxicol., 11 (1972) 415-432. 13 J.M. Ward and G. Vlahakis, Evaluation of hepatocellular neoplasms in mice, J. Natl. Cancer Inst., 61 (1978) 807-809.