TISSUE AND CELL, 199123 (5) 607-612 fQ 1991 Longman Group UK Ltd.
G. VERGANI”,
A. MAYERHOFERt
and A. BARTKES
ACUTE EFFECTS OF HUMAN GROWTH HORMONE ON LIVER CELLS IN VITRO: A COMPARISON WITH LIVERS OF MICE TRANSGENIC FOR HUMAN GROWTH HORMONE Keywords:
Human growth hormone,
transgenic
mice, rat liver cells culture, electron microscopy
ABSTRACT. We have examined the effects of human growth hormone (hGH), in concentrations comparable to those measured in plasma of transgenic mice expressing foreign GHs, on rat liver cells in culture. This treatment produced, within 24 and 48 hr, extreme heterogeneity in liver cell size, enlargement of nuclei, increase in the numbers of large nucleoli and nuclear protrusions, as well as appearance of numerous lipid droplets and accumulation of glycogen. These changes most likely indicate massive metabolic alterations and resemble changes present in oiuo in the livers of mice transgenic for hGH and other foreign GHs. Since morphological alterations in oitro were apparent within 24 hr. we conclude that GH acutely and directly affects liver cell morphology and function in vim and that the pathological lesions in oivo in the livers of transgenic mice are very likely a consequence of GH action.
Introduction
Various effects of the expression of foreign growth hormone (GH) genes on the morphology of the liver in transgenic mice have previously been described (Orian etaf., 1989; Quaife et al., 1989). The animals studied were transgenic for either ovine (0) GH, bovine (b) GH or human growth hormone releasing factor (hGRF), which elevates endogenous (mouse) GH secretion, fused to the mouse metallothionein I promoter. These genes were expressed mainly in kidney and liver. From these studies, it is known that pathological lesions of the livers of transgenic animals bearing GH genes or the GRF gene include abnormally large cell and nuclear size, intranuclear inclusions and extramedullary hematopoiesis. Interestingly, these
*Abteilung Anatomie, tAbteilung Anatomie and Zellbiologie, Universitlt Ulm, 7900 Ulm, Germany and *Department of Physiology, School of Medicine, Southern Illinois University, Carbondale, Illinois. 62901, USA. Address correspondence to: Dr. G. Vergani, Abteihmg Anatomie, Universitlt Ulm, Albert-Einstein-Allee 11, D-7900 Ulm, Germany. Received 12 April 1991 Revised 23 May 1991.
studies revealed that pathological lesions were present in the livers of mice expressing GHs but not human insulin-like growth factor-I (IGF-I) with the same promoter (Quaife et al., 1989). The reasons for these differences remain unknown. Possibilities include direct effects of GH on the liver cell, versus involvement of IGF-I in this process, or alternatively insertional mutagenesis. Furthermore, the question arises, whether the alterations in the livers of transgenic mice are a consequence of a slow developmental process or could be caused rapidly by GH. Therefore, we have examined the effect of human GH on the morphology of liver cells isolated from the rat at the light and electron microscope level, and have compared the findings with the histological appearance of the livers of transgenic mice expressing the gene for hGH.
Materials and Methods
mice were obtained from a line derived from animals generously provided by Dr. Thomas E. Wagner and described previously (Bartke et al., 1988; Selden et al., 1989). Livers were fixed in Bouin’s fixative, embedded into paraffin and sections were cut
Transgenic
VERGANI ET AL.
608
and stained with hematoxilin-eosin (H.E.). Rat liver parenchymal cells were prepared from a 20-day-old male rat (SIV 50, IVANOVAS , Kissleg, Germany), according to the procedure described by Vergani et al. (1979). Cells were grown as monolayers in Ham’s F-10 medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum and antibiotics in COz-incubator (5% CO2 and 95% air) at 37°C. Medium was changed every 3 days. Human growth hormone (courtesy of the National Hormone and Pituitary Program, Baltimore, MD, USA) was added to the medium (10 ng/ml) for 24 and 48 hr. This GH concentration was chosen to match the range of hGH in uiuo in hGH-transgenic mice, which ranged from about 3 to about 15 ng/ml serum (hGH,
Bartke et al., 1988; Selden et al., 1989; or 11.3 ng/ml, in bGH; cf. Mayerhofer et al., 1990). After that time, the cells were fixed and used for light or transmission electron microscopy (TEM) as described by Vergani and Frosch (1987). Results Morphology of the liver in mice transgenic for the hGH gene
Similarly to previous observations on the livers of transgenic mice expressing bGH or oGH, the histological picture of livers from mice transgenic for hGH showed enlarged hepatocytes, irregular nuclear membrane, increased members of nucleoli or fragments of nucleoli and disorganized bile ducts (Fig. 1). .,.
i c f3
4
Fig. 1. Liver of a control mouse (a) and liver of a transgenic mouse (b) expressing hGH. Note differences in cell sizes and nuclear sizes. Central vene (asterisk). bile duct (arrow). nucleus with inclusions (arrowhead). H.E.-staining. x 170.
EFFECTS OF GROWTH
FAmOR
ON LIVER CELLS
Fig. 2. Liver cell culture grown 24 hr in basal medium (control; a) and 24 hr with 10 ng/ml hGH (b). Note enlarged cell size in the treated group. x220.
Effects of hGH on morphology cultures
of liver cell
After 24 hr of exposure to hGH, liver cells revealed a marked heterogeneity of cell and nuclear size at the light and electron microscopic levels as compared to untreated controls. After 48 hr, these changes were even more pronounced. Alterations at the light microscope level included cytoplasmic blebbing and pronounced vacuolization (Fig. 2). Most strikingly, the diameter of cells ranged from 62-5 pm-112,5 pm, compared to 37.5 pm-62.5 pm in controls. Autofluoresence of H.E. stained cells (at wavelength 436 pm) clearly revealed the central nucleus with one or several nucleoli in control cells contrasting with unclear structure and seemingly empty introcytoplasmic spaces, resembling large vacuoles in hGH treated cells (Fig. 3). Electron microscopy showed in addition that the treated cells were characterized by accumulation of conspicuous lipid droplets (Fig. 4). Inclusions (resembling secondary lysosomes), vacuolization of the cytoplasm, damaged mitochondria, and apparently normal nuclear membrane (Fig. 4) were also observed in the hGH-exposed cells at the ultrastructural level.
Discussion It is well established that GH binds to its receptor on liver cells (cf Scott et al., 1985). Subsequently, the production of somatomedins (IGF I and II) by liver cells is induced. Somatomedins mediate the effects of GH on various organs (review by Baxter, 1988). However, GH itself is known to have additional effects on liver cell metabolism. For example, the incorporation of amino acids and nucleic acids into the liver cells is promoted by GH directly (Jefferson and Korner, 1967). In the present study we have not examined biochemical aspects of liver cells function, but rather liver cell morphology. However, increased numbers of lipid droplets, massive glycogen accumulation and most strikingly increased liver cell size in hGH treated cultures, indicate effects of GH on liver cell function. Very interestingly, these changes occur in vitro within 24 hr and are very similar to alterations found in vivo in the livers of mice expressing genes for various foreign GHs. Thus, previous studies and the present study have shown that hepatocellularmegally and pseudonuclear inclusions are striking features of the livers of transgenic mice expressing the genes
VERGANI ET AL
Fig. 3. Autofluorescence of liver cell culture grown 24 hr without GH (control; a) and 24 hr with 10 ng/ml hGH (b). Note mitosis (arrow) and cells with clear nucleus-cytoplasma border in (a). In contrast, note in (b) large cells, unclear nuclear structure and seemingly empty spaces, likely to present vacuoles (arrowhead). X260.
Fig. 4. Liver cell culture grown 24 hr without hGH (control; a) and 24 hr with 10 rig/ml hGH (b). Note that hGH treated cell contains electron dense lipid droplets, inclusions (I) resembling secondary lysosomes and mitochondria (arrows) with unclear structures. X11.250.
EFFECTS OF GROWTH
611
FACTOR ON LIVER CELLS
for oGH (Orian et al., 1989), bGH (Quaife et al., 1989), hGRF (Quaife et al, 1989) and hGH (this study). Comparable changes are conspicuously absent in transgenic mice expressing the IGF-I gene (Quaife et al., 1989). It should be mentioned that in contrast to other GHs, hGH in rodents also binds to the liver cell prolactin receptor (Amit et al., 1984) but in view of the effects of oGH, bGH and hGRF in uiuo, this mechanism is very unlikely to be involved. Taken together, these data from the study of livers of transgenic mice suggest that the effects of GH on liver cells are direct and not mediated via IGF I. However, they do not provide additional information on the ethiology of these pathological lesions. All transgenic mice examined expressed GH mainly in liver and kidney, due to the coupling of the GH genes with the mouse metallothionien I promoter region. Thus, insertional mutagenesis or effects exerted by GH before it leaves the cells, could be responsible for the lesions. -The results of the present study reveal, for the first time, that exogenous GH can produce comparable lesions by acting directly on liver cells in uitro. We furthermore show that the onset of the damages is acute and caused by concentrations of GH which fall into the range of GH measured in transgenic mice (Bartke et al., 1988; Selden et al. , 1989; see also Mayerhofer et al. , 1990). It is therefore conceivable that acute GH
actions may be responsible for the lesions in the livers of transgenic animals in uiuo. However, in uiuo the livers may have mechanisms to counteract GH effects more effectively than in uitro. Nevertheless, premature aging and death in transgenic mice are common (Bartke et al., 1988) and altered liver functions can be suspected to contribute to the premature aging. To summarize, in the present study we describe severe acute morphological alterations of rat liver cells as the consequences of exposure to exogenous hGH. The acute onset of the lesions is surprising and indicates that similar pathological lesions in the livers of transgenic mice are likely due to direct GH action. Altered liver cell function can be expected to occur concomitantly with morphological changes. Nowadays GH is widely applied especially in animal industry. Our present data might be viewed as an example that the use of GH bears unforseeable risks and therefore calls for caution when applying GH. Acknowledgements
The gift of human GH by the National Hormone and Pituitary Program, Baltimore, MD, USA is gratefully acknowledged. We thank Mrs. I. Urban and S. Amthor for expert technical assistance and Mrs. I. AlAynein for typing of the manuscript.
References Amit, T., Barkey, R. J., Gavish, M. and Youdim, M. B. 1984. Induction of prolactin (PRL) receptors by PRL in the rat lung and liver. Demonstration and characterization on a soluble receptor. Endocrinology, 114, 545-552. Bartke, A., Steger, R. W., Hodges, S. L., Parkening, T. A., Collins, T. J., Yun, J. S. and Wagner, T. E., 1988. Infertility in transgenic female mice with human growth hormone expression: Evidence for luteal failure. J. Exp. Zool., 248, 121-124. Baxter, R. C. 1988. The insulin-like growth factors and their binding protein. Camp. Biochem. PhysioL, 91B, 229 235. Jefferson, L. S. and Korner, A. 1967. A direct effect of growth hormone on the incorporation of precursors into proteins and nucleic acids of perfused rat liver. Biochem. J., 104, 826832. Mayerhofer, A., Weis, J., Bartke, A., Yun, J. S. and Wagner, T. E. 1990. Effects of trangenes for human and bovine growth hormone on age-related changes in ovarian morphology in mice. Amt. Rec., 227, 175-186. Orian. J. M., Lee, C. S., Weiss, L. M. and Brandon, M. R. 1989. The expression of a metallothionein-ovine growth hormone fusion gene in transgenic mice does not impair fertility but results in pathological lesions in the liver. Endocrinology,
124, 455-463.
Quaife, C. J., Mathews. L. S., Pinkert, C. A., Hammer, R. E., Brinster, R. L. and Palmiter, R. D. 1989. Histopathology associated with elevated levels of growth hormone and insulin-like growth factor I in transgenic mice. Endocrinology, 124, 40-48. Scott, C., Martin, J. L. and Baxter, R. C. 1985. Rat hepatocytes insulin-like growth factor I and binding protein: Effect of growth hormone in vitro and in viva. Endocrinology, 116, 1102-1107.
612
VERGANI ET AL.
Selden, R. F., June, J. S., Moore, D. D., Rowe, M:E., Maha, M. A., Wagner, T. E. and Goodman, H. M. 1989. Glucocorticoid regulation of human growth hormone expression in transgenic mice and transiently transfected cells. J. Endocrinol., 122, 49-60. Vergani, G., Pentz, S. and Herrmann, M. 1979. Effects of sera from scalded rats on epithelial liver cells in vitro. Virchows Arch. (B) Cell Path., 31, 31-35. Vergani, G. and Frosch, D. 1987. Morphologie epitheloider Rattenleberzellen in Kultur. Verb. Amt. Go., 81, 621622.
G. VERGANI”,
A. MAYERHOFERt
and A. BARTKES
ACUTE EFFECTS OF HUMAN GROWTH HORMONE ON LIVER CELLS IN VITRO: A COMPARISON WITH LIVERS OF MICE TRANSGENIC FOR HUMAN GROWTH HORMONE Keywords:
Human growth hormone,
transgenic
mice, rat liver cells culture, electron microscopy
ABSTRACT. We have examined the effects of human growth hormone (hGH), in concentrations comparable to those measured in plasma of transgenic mice expressing foreign GHs, on rat liver cells in culture. This treatment produced, within 24 and 48 hr, extreme heterogeneity in liver cell size, enlargement of nuclei, increase in the numbers of large nucleoli and nuclear protrusions, as well as appearance of numerous lipid droplets and accumulation of glycogen. These changes most likely indicate massive metabolic alterations and resemble changes present in oiuo in the livers of mice transgenic for hGH and other foreign GHs. Since morphological alterations in oitro were apparent within 24 hr. we conclude that GH acutely and directly affects liver cell morphology and function in vim and that the pathological lesions in oivo in the livers of transgenic mice are very likely a consequence of GH action.
Introduction
Various effects of the expression of foreign growth hormone (GH) genes on the morphology of the liver in transgenic mice have previously been described (Orian etaf., 1989; Quaife et al., 1989). The animals studied were transgenic for either ovine (0) GH, bovine (b) GH or human growth hormone releasing factor (hGRF), which elevates endogenous (mouse) GH secretion, fused to the mouse metallothionein I promoter. These genes were expressed mainly in kidney and liver. From these studies, it is known that pathological lesions of the livers of transgenic animals bearing GH genes or the GRF gene include abnormally large cell and nuclear size, intranuclear inclusions and extramedullary hematopoiesis. Interestingly, these
*Abteilung Anatomie, tAbteilung Anatomie and Zellbiologie, Universitlt Ulm, 7900 Ulm, Germany and *Department of Physiology, School of Medicine, Southern Illinois University, Carbondale, Illinois. 62901, USA. Address correspondence to: Dr. G. Vergani, Abteihmg Anatomie, Universitlt Ulm, Albert-Einstein-Allee 11, D-7900 Ulm, Germany. Received 12 April 1991 Revised 23 May 1991.
studies revealed that pathological lesions were present in the livers of mice expressing GHs but not human insulin-like growth factor-I (IGF-I) with the same promoter (Quaife et al., 1989). The reasons for these differences remain unknown. Possibilities include direct effects of GH on the liver cell, versus involvement of IGF-I in this process, or alternatively insertional mutagenesis. Furthermore, the question arises, whether the alterations in the livers of transgenic mice are a consequence of a slow developmental process or could be caused rapidly by GH. Therefore, we have examined the effect of human GH on the morphology of liver cells isolated from the rat at the light and electron microscope level, and have compared the findings with the histological appearance of the livers of transgenic mice expressing the gene for hGH.
Materials and Methods
mice were obtained from a line derived from animals generously provided by Dr. Thomas E. Wagner and described previously (Bartke et al., 1988; Selden et al., 1989). Livers were fixed in Bouin’s fixative, embedded into paraffin and sections were cut
Transgenic
VERGANI ET AL.
608
and stained with hematoxilin-eosin (H.E.). Rat liver parenchymal cells were prepared from a 20-day-old male rat (SIV 50, IVANOVAS , Kissleg, Germany), according to the procedure described by Vergani et al. (1979). Cells were grown as monolayers in Ham’s F-10 medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum and antibiotics in COz-incubator (5% CO2 and 95% air) at 37°C. Medium was changed every 3 days. Human growth hormone (courtesy of the National Hormone and Pituitary Program, Baltimore, MD, USA) was added to the medium (10 ng/ml) for 24 and 48 hr. This GH concentration was chosen to match the range of hGH in uiuo in hGH-transgenic mice, which ranged from about 3 to about 15 ng/ml serum (hGH,
Bartke et al., 1988; Selden et al., 1989; or 11.3 ng/ml, in bGH; cf. Mayerhofer et al., 1990). After that time, the cells were fixed and used for light or transmission electron microscopy (TEM) as described by Vergani and Frosch (1987). Results Morphology of the liver in mice transgenic for the hGH gene
Similarly to previous observations on the livers of transgenic mice expressing bGH or oGH, the histological picture of livers from mice transgenic for hGH showed enlarged hepatocytes, irregular nuclear membrane, increased members of nucleoli or fragments of nucleoli and disorganized bile ducts (Fig. 1). .,.
i c f3
4
Fig. 1. Liver of a control mouse (a) and liver of a transgenic mouse (b) expressing hGH. Note differences in cell sizes and nuclear sizes. Central vene (asterisk). bile duct (arrow). nucleus with inclusions (arrowhead). H.E.-staining. x 170.
EFFECTS OF GROWTH
FAmOR
ON LIVER CELLS
Fig. 2. Liver cell culture grown 24 hr in basal medium (control; a) and 24 hr with 10 ng/ml hGH (b). Note enlarged cell size in the treated group. x220.
Effects of hGH on morphology cultures
of liver cell
After 24 hr of exposure to hGH, liver cells revealed a marked heterogeneity of cell and nuclear size at the light and electron microscopic levels as compared to untreated controls. After 48 hr, these changes were even more pronounced. Alterations at the light microscope level included cytoplasmic blebbing and pronounced vacuolization (Fig. 2). Most strikingly, the diameter of cells ranged from 62-5 pm-112,5 pm, compared to 37.5 pm-62.5 pm in controls. Autofluoresence of H.E. stained cells (at wavelength 436 pm) clearly revealed the central nucleus with one or several nucleoli in control cells contrasting with unclear structure and seemingly empty introcytoplasmic spaces, resembling large vacuoles in hGH treated cells (Fig. 3). Electron microscopy showed in addition that the treated cells were characterized by accumulation of conspicuous lipid droplets (Fig. 4). Inclusions (resembling secondary lysosomes), vacuolization of the cytoplasm, damaged mitochondria, and apparently normal nuclear membrane (Fig. 4) were also observed in the hGH-exposed cells at the ultrastructural level.
Discussion It is well established that GH binds to its receptor on liver cells (cf Scott et al., 1985). Subsequently, the production of somatomedins (IGF I and II) by liver cells is induced. Somatomedins mediate the effects of GH on various organs (review by Baxter, 1988). However, GH itself is known to have additional effects on liver cell metabolism. For example, the incorporation of amino acids and nucleic acids into the liver cells is promoted by GH directly (Jefferson and Korner, 1967). In the present study we have not examined biochemical aspects of liver cells function, but rather liver cell morphology. However, increased numbers of lipid droplets, massive glycogen accumulation and most strikingly increased liver cell size in hGH treated cultures, indicate effects of GH on liver cell function. Very interestingly, these changes occur in vitro within 24 hr and are very similar to alterations found in vivo in the livers of mice expressing genes for various foreign GHs. Thus, previous studies and the present study have shown that hepatocellularmegally and pseudonuclear inclusions are striking features of the livers of transgenic mice expressing the genes
VERGANI ET AL
Fig. 3. Autofluorescence of liver cell culture grown 24 hr without GH (control; a) and 24 hr with 10 ng/ml hGH (b). Note mitosis (arrow) and cells with clear nucleus-cytoplasma border in (a). In contrast, note in (b) large cells, unclear nuclear structure and seemingly empty spaces, likely to present vacuoles (arrowhead). X260.
Fig. 4. Liver cell culture grown 24 hr without hGH (control; a) and 24 hr with 10 rig/ml hGH (b). Note that hGH treated cell contains electron dense lipid droplets, inclusions (I) resembling secondary lysosomes and mitochondria (arrows) with unclear structures. X11.250.
EFFECTS OF GROWTH
611
FACTOR ON LIVER CELLS
for oGH (Orian et al., 1989), bGH (Quaife et al., 1989), hGRF (Quaife et al, 1989) and hGH (this study). Comparable changes are conspicuously absent in transgenic mice expressing the IGF-I gene (Quaife et al., 1989). It should be mentioned that in contrast to other GHs, hGH in rodents also binds to the liver cell prolactin receptor (Amit et al., 1984) but in view of the effects of oGH, bGH and hGRF in uiuo, this mechanism is very unlikely to be involved. Taken together, these data from the study of livers of transgenic mice suggest that the effects of GH on liver cells are direct and not mediated via IGF I. However, they do not provide additional information on the ethiology of these pathological lesions. All transgenic mice examined expressed GH mainly in liver and kidney, due to the coupling of the GH genes with the mouse metallothionien I promoter region. Thus, insertional mutagenesis or effects exerted by GH before it leaves the cells, could be responsible for the lesions. -The results of the present study reveal, for the first time, that exogenous GH can produce comparable lesions by acting directly on liver cells in uitro. We furthermore show that the onset of the damages is acute and caused by concentrations of GH which fall into the range of GH measured in transgenic mice (Bartke et al., 1988; Selden et al. , 1989; see also Mayerhofer et al. , 1990). It is therefore conceivable that acute GH
actions may be responsible for the lesions in the livers of transgenic animals in uiuo. However, in uiuo the livers may have mechanisms to counteract GH effects more effectively than in uitro. Nevertheless, premature aging and death in transgenic mice are common (Bartke et al., 1988) and altered liver functions can be suspected to contribute to the premature aging. To summarize, in the present study we describe severe acute morphological alterations of rat liver cells as the consequences of exposure to exogenous hGH. The acute onset of the lesions is surprising and indicates that similar pathological lesions in the livers of transgenic mice are likely due to direct GH action. Altered liver cell function can be expected to occur concomitantly with morphological changes. Nowadays GH is widely applied especially in animal industry. Our present data might be viewed as an example that the use of GH bears unforseeable risks and therefore calls for caution when applying GH. Acknowledgements
The gift of human GH by the National Hormone and Pituitary Program, Baltimore, MD, USA is gratefully acknowledged. We thank Mrs. I. Urban and S. Amthor for expert technical assistance and Mrs. I. AlAynein for typing of the manuscript.
References Amit, T., Barkey, R. J., Gavish, M. and Youdim, M. B. 1984. Induction of prolactin (PRL) receptors by PRL in the rat lung and liver. Demonstration and characterization on a soluble receptor. Endocrinology, 114, 545-552. Bartke, A., Steger, R. W., Hodges, S. L., Parkening, T. A., Collins, T. J., Yun, J. S. and Wagner, T. E., 1988. Infertility in transgenic female mice with human growth hormone expression: Evidence for luteal failure. J. Exp. Zool., 248, 121-124. Baxter, R. C. 1988. The insulin-like growth factors and their binding protein. Camp. Biochem. PhysioL, 91B, 229 235. Jefferson, L. S. and Korner, A. 1967. A direct effect of growth hormone on the incorporation of precursors into proteins and nucleic acids of perfused rat liver. Biochem. J., 104, 826832. Mayerhofer, A., Weis, J., Bartke, A., Yun, J. S. and Wagner, T. E. 1990. Effects of trangenes for human and bovine growth hormone on age-related changes in ovarian morphology in mice. Amt. Rec., 227, 175-186. Orian. J. M., Lee, C. S., Weiss, L. M. and Brandon, M. R. 1989. The expression of a metallothionein-ovine growth hormone fusion gene in transgenic mice does not impair fertility but results in pathological lesions in the liver. Endocrinology,
124, 455-463.
Quaife, C. J., Mathews. L. S., Pinkert, C. A., Hammer, R. E., Brinster, R. L. and Palmiter, R. D. 1989. Histopathology associated with elevated levels of growth hormone and insulin-like growth factor I in transgenic mice. Endocrinology, 124, 40-48. Scott, C., Martin, J. L. and Baxter, R. C. 1985. Rat hepatocytes insulin-like growth factor I and binding protein: Effect of growth hormone in vitro and in viva. Endocrinology, 116, 1102-1107.
612
VERGANI ET AL.
Selden, R. F., June, J. S., Moore, D. D., Rowe, M:E., Maha, M. A., Wagner, T. E. and Goodman, H. M. 1989. Glucocorticoid regulation of human growth hormone expression in transgenic mice and transiently transfected cells. J. Endocrinol., 122, 49-60. Vergani, G., Pentz, S. and Herrmann, M. 1979. Effects of sera from scalded rats on epithelial liver cells in vitro. Virchows Arch. (B) Cell Path., 31, 31-35. Vergani, G. and Frosch, D. 1987. Morphologie epitheloider Rattenleberzellen in Kultur. Verb. Amt. Go., 81, 621622.
