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Correlation of liver tumor morphology and weight to incidence of pulmonary metastases in the mouse
113
Toxicology Letters, 7 (1980) 113-118 o Elsevier/North-Holland Biomedical Press
CORRELATION OF LIVER TUMOR MORPHOLOGY AND WEIGHT TO INCIDENCE OF PULMONARY METASTASES IN THE MOUSE
C.H. FRITH*, K.P. BAETCKE**,
C.J. NELSON***
and G. SCHIEFERSTEIN***
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 72211; and ***Department of Health, Education and Welfare, Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079 (U.S.A.) (Received May 30th, 1980) (Accepted July 8th, 1980)
SUMMARY
Benzidine dihydrochloride administered in water at 0, 30, 60, 120, 200 or 400 ppm to 1664 mice induced a total of 368 hepatocellular carcinomas, of which 34 or 9.2% metastasized to the lungs. Liver tumors which were larger and heavier were more prone to metastasize and all that metastasized contained areas of prominent trabecular formation.
INTRODUCTION
Recent studies have shown that the incidence of pulmonary metastases from hepatocellular carcinomas in the mouse is higher than previously expected [3-51. We have attempted to relate the incidence of pulmonary metastases of hepatocellular carcinomas in mice to the tumorous liver weight and to the morphology of the liver tumor. MATERIALS
AND METHODS
1664 Fi (C57BL/GJC3Hf/Nctr females X BALB/cStCrlC3H/Nctr males) and Fz (F, females X Fi males) mice, after weaning, were housed, 4 per cage, in a room maintained at 22-2’4°C. Food and water were provided ad lib. The animals received Purina 5OlOC meal and were dosed with water containing 0, 30,60, 120, 200 and 400 parts per million (ppm) of benzidine dihydrochloride (Allied Chemical Co., Buffalo, NY). Groups of mice were killed at 40,60 and 80 weeks for pathologic study [1]. *Reprint requests to Dr. Frith at Pathology Services Project, NCTR. Abbreviation:
CID, carcass identification.
114
Each animal that died was given a CID number and routine necropsies were performed. All livers were weighed except those of mice that died. Gross and microscopic findings were collected on 45 tissues or organs per mouse as described by Erith et al. [2] . Sections of grossly visible lesions and of the median, left lateral and right lateral lobes of the liver were collected for microscopic evaluation. The tissues were fixed in Bouin’s solution for 18-24 h. After fixation the tissues were routinely trimmed, placed in plastic cassettes for processing on an autotechnicon (4-h cycle) and embedded in paraffin blocks. Routine paraffin sections were prepared, cut at 5 pm and stained with hematoxylin and eosin (H & E) [ 21. A single section of all lobes of the lungs was examined. RESULTS
Detailed results of this study, including the incidence of hepatocellular neoplasms and of pulmonary metastases are reported in another paper [l] . The purpose of the present contribution is to attempt to correlate tumorous liver weight and morphology with the incidence of pulmonary metastases. Since the first pulmonary metastasis was seen on day 366 of the study, all animals that died or were killed prior to day 366 were deleted from the analyses. The analysis of covariance, adjusting for final body weight, revealed no significant differences between the liver weight for the F, and Fz mice, which were therefore combined. A total of 368 hepatocell~~ carcinomas were seen in this study and 34 or 9.2% metastasized to the lungs. The incidence of pulmonary me&stases was similar in both sexes. All hepatocellular carcinomas which metastasized to the lungs (Fig. 1) contained areas of trabecular formation in the primary site (Fig. 2). 27 of the 34 animals with pulmonary metastases were moribund and their livers were weighed. Liver weights with and without liver tumors were adjusted for body weight. The adjusted mean liver weights in animals with no hepatocellular carcinoma were 1577 mg and 1201 mg in the controls, and 1457 mg and 1216 mg in treated males and females, respectively (Table I). The difference in liver weights between the males and females was significant in both groups (P < 0.05). The difference was not significant between the control and treated mice without hepatocellul~ carcinoma. The adjusted liver weights in the male and female treated animals with hepatocellular carcinoma and no pulmonary me&stases were 2736 and 3903 mg, and in male and female treated mice with hepatocellular carcinoma and pulmonary metastastes were 4886 and 5298 mg, respectively (Table I). The mean adjusted liver weight of mice with pulmonary metastases was increased significantly (P < 0.05) in both sexes. The association between tumorous liver weight and pulmonary metastases is also presented in Fig. 3. Only one pulmonary metastasis (4%) occurred with tumorous livers weighing < 1500 mg and 89% occurred with tumorous livers weighing > 3000 mg.
115
Fig. 1. Metastatic
hepatocellular
carcinoma.
H 8z E X 350.
TABLE I CORRELATION OF ADJUSTED PULMONARY ME’I’ASTASES
MEAN LIVER AND/OR
LIVER TUMOR WEIGHT WITH
Groups
Sex
Number of animals
Adjusted mean liver weight -I S.D. (mg) (includes tumor weight if present)
(a) Control animals without hepatoceIIuiar carcinoma
M F
179 183
1577 * 249 1201 f 188
(b) Treated animals without hepatocellular carcinoma
M F
681 360
1466 + 189 1216 + 274
(c) Treated animals with hepatocelIuIar carcinoma and no metastaser
M F
75 259
2603 f 1301 3903 f 1907
(d) Treated animals with
M F
6 21
4386 f 1423 5298 f 2163
hepatocellular carcinoma and pulmonary metastases
116
Fig. 2. Prominent trabecular formation associated with metastases. H & E
1500-3000
3000-4500
her Tumor Wewht IMKI
=-4X10
X
350.
117 DISCUSSION
Of a total of 1664 animals, 34 of 368 (9.2%) hepatocellular carcinomas metastasized to the lungs. The incidence of pulmonary metastases might have been higher if additional sections of the lungs had been examined. Exposure to benzidine dihydrochloride resulted in a slight reduction below normal in the weight of the livers of male mice until liver neoplasms began to develop. The weights of the liver tumors which metastasized to the lungs were significantly higher in both sexes compared with the weights of the liver tumors which did not metastasize. The incidence of pulmonary metastases was also related to the degree of differentiation and the moderately well and poorly differentiated hepatocellular carcinomas metastasized more frequently than the well differentiated hepatocellular carcinomas [l] . The mitotic index of liver tumors in this study was not evaluated, but Ward et al. [6] reported that the number of mitotic figures per high-power field was not related to the size or the metastatic capability of hepatocellular carcinomas in mice. The fact that the pulmonary metastases were relatively rare until the 80week serial killing [l] indicates that the liver tumors must reach a large weight before the metastases occur. Prominent trabecular formation was also a characteristic of all hepatocellular carcinomas which metastasized. Vesselinovitch et al. [ 51 have also demonstrated that hepatocellular carcinomas with a trabecular pattern are more likely to metastasize. Ward et al. [6] reported that prominent trabeculae were seen in relatively few small liver tumors, whereas prominent trabecular formation was a common characteristic of large hepatocellular tumors. In summary, trabecular formation was a common finding of metastatic hepatocellular carcinomas in mice treated with benzidine dihydrochloride. Since trabecular formation appears to be more common in heavier tumorous livers and since malignant tumors are significantly heavier, it follows that the incidence of metastases correlates with the weight of hepatocellular carcinomas and would be higher in heavier tumors.
Fig. 3. Correlation of liver tumor weight to incidence of pulmonary metastases. *Actual number of metastases is represented by number in bottom of each bar. Number on top of bar represents % metastases for respective liver tumor weight.
118 REFERENCES 1 C.H. Frith, K.P. Baetcke, C.J. Nelson and G. Schieferstein, Importance of mouse liver tumors in carcinogenesis bioassay studies using benzidine dihydrochloride as a model, Toxicol. Lett., 4 (1979), 507-518. 2 C.H. Frith, B. Highman and A.J. Konvicka, Advances in automation for experimental pathology, Lab, Animal Sci., 26 (1976) 171-185. 3 K.L. Hoover, S.F. Stinson and J.M. Ward, Histopathological differences between nitrogen-induced and naturally occurring hepatocellular carcinomas in the B6C3F, mouse, Abstract Lab. Invest., 40 (1979) 26. 4 A.P. Kyriazis, M. Koda and S.D. Vesselinovitch, Metastatic rate of liver tumors induced by diethyinitrosamine in mice, Cancer Res., 34 (1974) 2881-2886. 5 S.D. Vesselinovitch, N. Mihailovich and K.V.N. Rao, Morphology and metastatic nature of induced hepatic nodular lesions in C57BLXC3H F, mice, Cancer Res., 38 (1978) 2003-2010. 6 J.M. Ward and G. Vlahakis, Evahzation of hepatocellular neoplasms in mice, J. Natl. Cancer Inst., 61(1978) 807-811
Toxicology Letters, 7 (1980) 113-118 o Elsevier/North-Holland Biomedical Press
CORRELATION OF LIVER TUMOR MORPHOLOGY AND WEIGHT TO INCIDENCE OF PULMONARY METASTASES IN THE MOUSE
C.H. FRITH*, K.P. BAETCKE**,
C.J. NELSON***
and G. SCHIEFERSTEIN***
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 72211; and ***Department of Health, Education and Welfare, Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079 (U.S.A.) (Received May 30th, 1980) (Accepted July 8th, 1980)
SUMMARY
Benzidine dihydrochloride administered in water at 0, 30, 60, 120, 200 or 400 ppm to 1664 mice induced a total of 368 hepatocellular carcinomas, of which 34 or 9.2% metastasized to the lungs. Liver tumors which were larger and heavier were more prone to metastasize and all that metastasized contained areas of prominent trabecular formation.
INTRODUCTION
Recent studies have shown that the incidence of pulmonary metastases from hepatocellular carcinomas in the mouse is higher than previously expected [3-51. We have attempted to relate the incidence of pulmonary metastases of hepatocellular carcinomas in mice to the tumorous liver weight and to the morphology of the liver tumor. MATERIALS
AND METHODS
1664 Fi (C57BL/GJC3Hf/Nctr females X BALB/cStCrlC3H/Nctr males) and Fz (F, females X Fi males) mice, after weaning, were housed, 4 per cage, in a room maintained at 22-2’4°C. Food and water were provided ad lib. The animals received Purina 5OlOC meal and were dosed with water containing 0, 30,60, 120, 200 and 400 parts per million (ppm) of benzidine dihydrochloride (Allied Chemical Co., Buffalo, NY). Groups of mice were killed at 40,60 and 80 weeks for pathologic study [1]. *Reprint requests to Dr. Frith at Pathology Services Project, NCTR. Abbreviation:
CID, carcass identification.
114
Each animal that died was given a CID number and routine necropsies were performed. All livers were weighed except those of mice that died. Gross and microscopic findings were collected on 45 tissues or organs per mouse as described by Erith et al. [2] . Sections of grossly visible lesions and of the median, left lateral and right lateral lobes of the liver were collected for microscopic evaluation. The tissues were fixed in Bouin’s solution for 18-24 h. After fixation the tissues were routinely trimmed, placed in plastic cassettes for processing on an autotechnicon (4-h cycle) and embedded in paraffin blocks. Routine paraffin sections were prepared, cut at 5 pm and stained with hematoxylin and eosin (H & E) [ 21. A single section of all lobes of the lungs was examined. RESULTS
Detailed results of this study, including the incidence of hepatocellular neoplasms and of pulmonary metastases are reported in another paper [l] . The purpose of the present contribution is to attempt to correlate tumorous liver weight and morphology with the incidence of pulmonary metastases. Since the first pulmonary metastasis was seen on day 366 of the study, all animals that died or were killed prior to day 366 were deleted from the analyses. The analysis of covariance, adjusting for final body weight, revealed no significant differences between the liver weight for the F, and Fz mice, which were therefore combined. A total of 368 hepatocell~~ carcinomas were seen in this study and 34 or 9.2% metastasized to the lungs. The incidence of pulmonary me&stases was similar in both sexes. All hepatocellular carcinomas which metastasized to the lungs (Fig. 1) contained areas of trabecular formation in the primary site (Fig. 2). 27 of the 34 animals with pulmonary metastases were moribund and their livers were weighed. Liver weights with and without liver tumors were adjusted for body weight. The adjusted mean liver weights in animals with no hepatocellular carcinoma were 1577 mg and 1201 mg in the controls, and 1457 mg and 1216 mg in treated males and females, respectively (Table I). The difference in liver weights between the males and females was significant in both groups (P < 0.05). The difference was not significant between the control and treated mice without hepatocellul~ carcinoma. The adjusted liver weights in the male and female treated animals with hepatocellular carcinoma and no pulmonary me&stases were 2736 and 3903 mg, and in male and female treated mice with hepatocellular carcinoma and pulmonary metastastes were 4886 and 5298 mg, respectively (Table I). The mean adjusted liver weight of mice with pulmonary metastases was increased significantly (P < 0.05) in both sexes. The association between tumorous liver weight and pulmonary metastases is also presented in Fig. 3. Only one pulmonary metastasis (4%) occurred with tumorous livers weighing < 1500 mg and 89% occurred with tumorous livers weighing > 3000 mg.
115
Fig. 1. Metastatic
hepatocellular
carcinoma.
H 8z E X 350.
TABLE I CORRELATION OF ADJUSTED PULMONARY ME’I’ASTASES
MEAN LIVER AND/OR
LIVER TUMOR WEIGHT WITH
Groups
Sex
Number of animals
Adjusted mean liver weight -I S.D. (mg) (includes tumor weight if present)
(a) Control animals without hepatoceIIuiar carcinoma
M F
179 183
1577 * 249 1201 f 188
(b) Treated animals without hepatocellular carcinoma
M F
681 360
1466 + 189 1216 + 274
(c) Treated animals with hepatocelIuIar carcinoma and no metastaser
M F
75 259
2603 f 1301 3903 f 1907
(d) Treated animals with
M F
6 21
4386 f 1423 5298 f 2163
hepatocellular carcinoma and pulmonary metastases
116
Fig. 2. Prominent trabecular formation associated with metastases. H & E
1500-3000
3000-4500
her Tumor Wewht IMKI
=-4X10
X
350.
117 DISCUSSION
Of a total of 1664 animals, 34 of 368 (9.2%) hepatocellular carcinomas metastasized to the lungs. The incidence of pulmonary metastases might have been higher if additional sections of the lungs had been examined. Exposure to benzidine dihydrochloride resulted in a slight reduction below normal in the weight of the livers of male mice until liver neoplasms began to develop. The weights of the liver tumors which metastasized to the lungs were significantly higher in both sexes compared with the weights of the liver tumors which did not metastasize. The incidence of pulmonary metastases was also related to the degree of differentiation and the moderately well and poorly differentiated hepatocellular carcinomas metastasized more frequently than the well differentiated hepatocellular carcinomas [l] . The mitotic index of liver tumors in this study was not evaluated, but Ward et al. [6] reported that the number of mitotic figures per high-power field was not related to the size or the metastatic capability of hepatocellular carcinomas in mice. The fact that the pulmonary metastases were relatively rare until the 80week serial killing [l] indicates that the liver tumors must reach a large weight before the metastases occur. Prominent trabecular formation was also a characteristic of all hepatocellular carcinomas which metastasized. Vesselinovitch et al. [ 51 have also demonstrated that hepatocellular carcinomas with a trabecular pattern are more likely to metastasize. Ward et al. [6] reported that prominent trabeculae were seen in relatively few small liver tumors, whereas prominent trabecular formation was a common characteristic of large hepatocellular tumors. In summary, trabecular formation was a common finding of metastatic hepatocellular carcinomas in mice treated with benzidine dihydrochloride. Since trabecular formation appears to be more common in heavier tumorous livers and since malignant tumors are significantly heavier, it follows that the incidence of metastases correlates with the weight of hepatocellular carcinomas and would be higher in heavier tumors.
Fig. 3. Correlation of liver tumor weight to incidence of pulmonary metastases. *Actual number of metastases is represented by number in bottom of each bar. Number on top of bar represents % metastases for respective liver tumor weight.
118 REFERENCES 1 C.H. Frith, K.P. Baetcke, C.J. Nelson and G. Schieferstein, Importance of mouse liver tumors in carcinogenesis bioassay studies using benzidine dihydrochloride as a model, Toxicol. Lett., 4 (1979), 507-518. 2 C.H. Frith, B. Highman and A.J. Konvicka, Advances in automation for experimental pathology, Lab, Animal Sci., 26 (1976) 171-185. 3 K.L. Hoover, S.F. Stinson and J.M. Ward, Histopathological differences between nitrogen-induced and naturally occurring hepatocellular carcinomas in the B6C3F, mouse, Abstract Lab. Invest., 40 (1979) 26. 4 A.P. Kyriazis, M. Koda and S.D. Vesselinovitch, Metastatic rate of liver tumors induced by diethyinitrosamine in mice, Cancer Res., 34 (1974) 2881-2886. 5 S.D. Vesselinovitch, N. Mihailovich and K.V.N. Rao, Morphology and metastatic nature of induced hepatic nodular lesions in C57BLXC3H F, mice, Cancer Res., 38 (1978) 2003-2010. 6 J.M. Ward and G. Vlahakis, Evahzation of hepatocellular neoplasms in mice, J. Natl. Cancer Inst., 61(1978) 807-811