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Isolation and enrichment of cells resistant to iron accumulation from carcinogen-induced rat liver altered foci
EXPERIMENTAL
AND
MOLECULAR
Isolation Accumulation Naylor
Dana
PATHOLOGY
37, 101-110
(1982)
and Enrichment of Cells Resistant to Iron from Carcinogen-Induced Rat Liver Altered
HIDEKI
MORI,’
Institute
for
Received
Disease
December
BING Mu,~
Foci
AND GARY M. WILLIAMS
Prevention, American Health Valhalla, New York IO595 21, 1981, and in revised
form
Foundation,
April
1 Dana
Road.
20, 1982
Enrichment of cells from rat liver iron-excluding foci induced by 2-acetylaminofluorene was performed using density gradients. Rats were continuously fed a diet containing the carcinogen for 8 to 13 weeks to induce altered foci that were iron excluding and positive for y-glutamyl transpeptidase activity. At weekly intervals, and after loading with iron by subcutaneous injection, the livers were perfused with collagenase and dissociated. Fractionation of dissociated control and treated liver cells on Ficoll gradients yielded three cellular fractions. In preparations from carcinogen-exposed rats the percentage of hepatocytes excluding iron and positive for y-glutamyl transpeptidase was always higher in the top (least dense) fraction than in the other lower fractions or the original population of dissociated hepatocytes. Moreover, the total number of hepatocytes in the top fraction increased progressively with the duration of feeding. These observations indicate that iron-excluding hepatocytes of enzyme-altered foci are less dense than other hepatocytes and can be enriched by gradient centrifugation. The progressive increase in the number of these altered liver cells with continued carcinogen exposure is consistent with a role for these cells in the development of liver tumors.
INTRODUCTION The altered focus is a small lesion of abnormal hepatocytes which appears in rat liver soon after exposure to experimental hepatocarcinogens and is regarded as an important precursor lesion for hepatic nodules and carcinomas (Bannasch, 1976; Barbason and Betz, 1981; Emmelot and Scherer, 1980; Farber, 1980; Kitagawa, 1976; Pitot et al., 1978; Pitot and Sirica, 1980; Rabes and Szymkowiak, 1979; Scherer and Emmelot, 1976; Schulte-Herman et al., 1981; Williams, 1976, 1980). If cells from such foci could be isolated, they would be useful for a variety of biological and biochemical studies to better define the nature of early alterations in hepatocarcinogenesis. The property of exclusion of cellular iron was developed by Williams and colleagues as a marker for abnormal populations of liver cells, including altered foci (Williams and Yamamoto, 1972; Williams, 1976, 1980). Theoretically, this property could result in a difference in the density between iron-excluding cells from altered foci and iron-containing normal hepatocytes prepared from iron-loaded livers and could thus be useful for the isolation of the altered cells on density gradients. Equilibrium density centrifugation has been applied to suspensions of liver cells to isolate hepatocytes by a number of authors (Castagna and Chauveau, 1969; ’ Present address: Department of Pathology, Gifu University School of Medicine, 40 TsukasaMachi, Gifu City 500, Japan. 2 Visiting pathologist from Shanghai Medical College of the Ministry of Railways, Shanghai, People’s Republic of China. 3 To whom correspondence and requests for reprints should be sent. 101 0014-4800/82/040101-10$02.00/O Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Walter et al., 1973; Drochmans et al., 1975, Pretlow and Williams, 1975; Tulp et al., 1976; Horsfall and Ketterer, 1976). Among density gradient techniques, continuous gradients have been reported to be more effective than discontinuous ones (Pretlow et al., 1975; Karp et al., 1980). Pretlow and Williams (1975) also reported that velocity sedimentation was superior to isopycnic centrifugation for the separation of hepatocytes. In the present study, we attempted to isolate, by a velocity sedimentation method using continuous gradients of Ficoll, cells from iron-excluding enzymealtered foci induced in rat liver by feeding a diet containing 2-acetylaminofluorene (AAF). Following production of hepatic siderosis by iron injection, livers were dissociated by collagenase perfusion and single-cell suspension were fractionated. Iron-excluding cells from foci were found to sediment in less dense regions of the gradients than iron-containing hepatocytes and thus could be enriched. A progressive increase in the proportion of the iron-excluding population resulted from continued feeding of the carcinogen, indicating the importance of these altered cells in liver tumor development. MATERIALS
AND METHODS
Liver Cell Isolation Livers containing cells from enzyme altered foci were obtained from 18 male F344 rats (Charles River) which had been fed a diet containing 0.02% AAF for 8- 13 weeks, starting at 8 weeks of age. Four control rats were fed the basal diet NIH-07. In order to produce hepatic siderosis, rats were injected subcutaneously with 0.1 ml iron dextran (nonemic, Burns-Biotec) per 50 g body wt three times a week for 3 weeks prior to killing. Three rats were used for cell isolation every week for 6 weeks beginning after 8 weeks of carcinogen feeding. Liver dissociation was performed according to the procedures described by Williams et al. (1977). Briefly, the liver was perfused through the portal vein with 0.5 mM ethylene glycol bis(P-aminoethyl ether)-iV,N’-tetraacetic acid (EGTA; Sigma Chemical Co.) in Hanks’ Ca2+ + Mg 2+-free balanced salt solution (Flow Laboratories, Inc.) buffered with 0.01 M Hepes (Calbiochem-Behring Corp.), pH adjusted to 7.35 with 1 N NaOH for 4 min; and then perfused with 100 units/ml collagenase (Sigma, Type 1) in Williams’ Medium E (WME; Flow Laboratories, Inc. (Williams and Gunn, 1974)) buffered with 0.01 M Hepes, pH adjusted to 7.35 for 12 min. After perfusion, cells were detached by gentle combing with a stainless-steel round-tooth comb and transferred to 50-ml centrifuge tubes, pipetted, sedimented at 1OOgfor 5 min, and then resuspended in WME. This technique routinely provides a yield of 203 + 78 x lo6 cells per 100 g body wt with 79 +- 7% viability (Williams et al., 1978). The cell suspension was then incubated at 37°C with the addition of DNase (Sigma), 2.2 mg/ml phosphate-buffered saline (PBS; composition in g/liter: 8.0 NaCl, 0.20 KH2POI, 1.15 Na,HPOJ per 10 ml of cell suspension. This was followed by addition of protease (Type VI, Sigma), 5 mg/ml PBS per 10 ml of cell suspension for 15 min. After the incubation, the suspension was filtered with 25-pm Nitex. Viability was quantified by trypan blue exclusion. Density Gradients Linear density gradients of Ficoll400 (average molecular weight-400,000 Pharmacia Fine Chemicals) in PBS were constructed in 15-ml polycarbonate centrifuge
ENRICHMENT
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tubes with the use of a lo-ml-capacity two-chambered gradient maker (Buchler Corp.). The gradients were constructed with the use of 5 ml of 8% (w/w) Ficoll solutions and 5 ml of 35% (w/w) Ficoll solutions on top of a I.5ml cushion of 40% Ficoll. Refractive indices of the gradients were measured with a refractometer (Bausch and Lomb) and the linearity of the gradients was confirmed. Liver cell suspensions of 1.5 x IO6 cells were layered on top of the gradients. The gradients were then centrifuged (Sorvall GLC-2B) at room temperature at 96g for 20 min using a swinging bucket rotor. Following the centrifugation, a volume equal to that of the added cells was discarded from the top of each gradient and the remainder of the gradient was collected in four fractions from the top in volumes of 4, 3, 1.5, and 1.5 ml. Hepatocytes were located mainly in the lower third of the gradients. For removal of Ficoll, each fraction was resuspended in WME and centrifuged at 800 t-pm for 5 min. After sedimentation these were resuspended and counted, together with determination of viability by trypan blue exclusion. Aliquots of cells were smeared on glass slides and air-dried. The attached cells on slides were examined microscopically after staining for iron (Prussian blue) or y-glutamyl transpeptidase (GGT) by standard methods (Hirota and Williams, 1979). The cell diameter was measured with a microreticle attached to the microscope. RESULTS Cell Isolation Perfusion of the liver with collagenase destroyed the normal sinusoidal endothelial structure and trabecular pattern of hepatocytes, as previously described (Williams et al., 1977). Nevertheless, foci of GGT-positive, iron-excluding cells could still be identified (Figs. 1 and 2). In control rats, no GGT-positive periportal hepatocytes, as seen in older animals (Kitagawa et al., 1980), were present. The
FIG. 1. Postperfusion liver from a rat fed AAF for 8 weeks and then loaded with iron. A hepatocelIular population showing positive GGT activity is located in the periportal region. GGT stain, X 144.
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FIG. 2. Serial section of the liver illustrated in Fig. 1. The GGT-positive hepatocellular population is also resistant to iron accumulation, seen as dark reaction product in surrounding hepatocytes. Iron stain, X 144.
combing procedure used to detach hepatocytes leaves behind a fibrovascular connective tissue plug containing bile ducts (Laishes and Williams, 1976) whose cells are also GGT positive (Glenner et al., 1962). After the dissociation and Nitex filtration of cells, about 80% were present as single cells and the viability by trypan blue exclusion was approximately 95% in preparations from control rats and at all stages of AAF feeding. In preparations from control rats not exposed to AAF, no GGT-positive cells were present and over 9% of cells contained stainable iron. In preparations from carcinogen-exposed rats, diffuse or granular cytochemical activity of GGT was present in cells (Fig. 3), the proportion depending upon the duration of exposure. Likewise, iron-excluding cells were identified in these preparations (Fig. 4). Cell Separation
Of the four fractions collected from Ficoll gradients, the top fraction (4 ml) contained mainly cell debris, nuclei, red blood cells, and small bile duct cells. Almost all viable cells were found in the lower three fractions. The viability of cells in these three fractions was 60-70% and was similar in each fraction from both control and exposed rats at each interval of feeding. The total recovery (total living cells from three fractions/living cells layered) was 45- 55% and was also similar at all stages. Aggregated cells were generally seen in the bottom fraction. The proportion of viable cells in each fraction and its progressive change with feeding of AAF are shown in Fig. 5. The number of cells in upper fraction 2 gradually increased with the duration of AAF feeding while that of lower fraction 4 decreased.
ENRICHMENT
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FIG. 3. Isolated hepatocytes from fraction 2 from a rat given AAF for 9 weeks. Positive activity of GGT is present in several dark cells in the lower right. GGT stain, x575.
Enrichment
of Altered
Hepatocytes
The preparations from control rats yielded cell fractions that were greater than 9% iron containing and completely negative for GGT. For preparations from carcinogen-exposed rats, the percentage of altered single cells with GGT activity among the total single cells is shown in Fig. 6. The percentage of GGT-positive
FIG. 4. Isolated hepatocytes from fraction 2 from a rat given AAF for 9 weeks. Two cells in the upper left are deficient in iron accumulation. Iron stain, x575.
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FIG. 5. Proportion AAF feeding. Open
AND
WILLIAMS
OF AAF FEEDING
of viable hepatocytes in each fraction bars, fraction 2; stippled bars, fraction
and its progressive change 3; bars with oblique lines,
with stages of fraction 4.
cells in fraction 2 was always higher than in the other lower fractions (3 and 4) or in the original population of cells before the centrifugation. In particular, this separation was prominent in the early stages of AAF feeding; thus, the enrichment of GGT-positive cells in fraction 2 at 8 weeks was more than fourfold over that in fraction 4 and 3.5 times that of the original cell population. A similar enrichment occurred for iron-excluding altered cells (Fig. 7) (as iron-excluding cells, both those with complete absence of iron reaction and those with very weak reaction were counted). In general, the percentage of iron-resistant cells in each fraction was relatively higher than that of GGT-positive cells (compare Figs. 6 and 7). Characteristics
of Altered
Cells
The mean diameter of GGT-positive cells versus that of GGT-negative cells in each fraction is shown in Table I. The diameter of GGT-positive or -negative cells I4 -
. - FRACTION
WEEKS
FIG. 6. Percentage the dissociate. Mean
of GGT-positive value from three
OF MF
4
FEEDING
hepatocytes in the total rats at each week.
single
hepatocytes
of each fraction
or
ENRICHMENT
LP 0 6
OF
9
ALTERED
I II
IO WEEKS
LIVER
OF AAF
CELLS
12
107
.
I 13
FEEDING
FIG. 7. Percentage of iron-excluding hepatocytes in the total single hepatocytes of each fraction or the dissociate. Mean value from three rats at each week.
in the upper fraction was slightly less than that of the lower fractions and the diameter of GGT-positive cells increased gradually during carcinogen feeding. Generally, up to 10 weeks of AAF feeding, the mean diameter of GGT-positive cells was less than that of GGT-negative cells, but after the 10 weeks, the mean size exceeded that of negative cells, which were of the same size throughout the AAF feeding. DISCUSSION In this study, liver cells from AAF-exposed rats that were loaded with iron prior to killing were dissociated and separated on Ficoll gradients in an effort to enrich cells from iron-excluding altered foci. The total number of cells in the less dense upper fraction of the gradient increased during carcinogen feeding while that of the denser lower fractions decreased, and the percentage of iron-excluding or GGTpositive cells in the less dense top fraction was always higher than that in the lower fractions or in the initial dissociate. Size of GGT-Positive
TABLE I or -Negative Hepatocytes in Each Fraction
Diameter (pm) of GGT-positive Fraction 2 3 4
hepatocytes”
8 weeks
9 weeks
10 weeks
11 weeks
12 weeks
13 weeks
20.6 f 2.1’ 21.4 f 2.2 22.7 f 2.3
20.4 f 2.3 22.1 f 2.5 23.1 + 2.4
22.0 -c 2.5 22.8 + 2.6 24.0 -c 2.9
23.0 t 3. I 23.8 f 3.4 24.9 2 3.5
23.4 t 3.1 24.4 f 3.6 24.9 -c 3.7
23.5 + 3.5 25.1 f 3.6 26.0 k 3.2
Diameter (pm) of GGT-negative hepatocytes* 22.6 ‘- 3.0 23.9 f 3.1 24.4 f 3.2
a Mean value from three rats (100 random single cells/rat) at each week. b Mean value from total rats through 8- 13 weeks. ’ The difference in size of cells between each fraction of each interval of feeding is statistically significant (P < 0.05) except for fraction 3 compared to fraction 4 at 12 weeks. The difference in size of cells in fraction 2 at each interval of feeding is statistically significant except comparison between Week 8 and Week 9.
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The enriched iron-excluding, GGT-positive cells must be derived from the carcinogen-induced altered foci composed of cells with the same characteristics. They could not represent zonally GGT-positive cells seen in older animals (Kitagawa et al., 1980) since these were not present in the livers of the young rats used in this study and thus, no GGT-positive cells occurred in fractions from control rats. Moreover, such cells are not iron excluding, which represents one of the advantages of this property as a marker. Also, the enriched cells were too numerous and too large to be bile duct cells, which in any event are largely excluded from the cell preparations by the technique used. Thus, the present results demonstrate that iron-excluding cells from altered foci induced by AAF are relatively lighter than hepatocytes that accumulate cellular iron and can be enriched by taking advantage of their resistance to iron accumulation. The percentage of iron-excluding or GGT-positive altered cells in the upper fraction was particularly high in the early stages of carcinogen exposure. This seemed to be related to the size of the hepatocytes; i.e., the mean diameter of GGT-positive cells in the early stages was smaller than that in the later stages. Thus, separation of altered cells from hepatocytes could be most effective during the initial stages of appearance of altered foci. The percentage of iron-excluding cells in each fraction was generally greater than that of the corresponding GGT-positive cells. This may relate to the fact that the property of iron exclusion is more developed early in carcinogen administration than the enzyme histochemical abnormalities involving GGT, adenosine triphosphatase, and glucose 6-phosphatase (Hirota and Williams, 1979). Moreover, the present data show that the property of iron exclusion is also reliable as a marker for altered cells in dissociated single-cell preparations. Isolation of hepatocytes on density gradients has been reported by several laboratories (Castagna and Chauveau, 1969; Walter et al., 1973; Drochmans et al., 1975; Pretlow and Williams, 1975; Tulp et al., 1976; Horsfall and Ketterer, 1976). Recently, Doolittle and Richter (198 1) used differential centrifugations to separate Kupffer cells from hepatocytes. However, the isolation of altered cells from animals treated with hepatocarcinogens has not been extensively studied. Horsfall and Ketterer (1976) reported fractionation of isolated liver cells from rats treated with N,N-dimethyl-4-aminoazobenzene. Interestingly, they noted an increase in slowly sedimenting cells similar to the present findings, but could not identify these cells as originating in preneoplastic foci. Using GGT as a marker for altered cells, Jacobs et al. (1981) reported the enrichment of two GGT-positive populations from livers of ethionine-treated rats. One of these was suggested to represent “oval” cells and the other probably contained cells from foci. In the present study, enrichment of altered cells from foci was also obtained, but their complete purification was not achieved. Modification of the methods of density gradient isolation in combination with other methods, such as cytotoxic selection of cells from foci (Williams et al., 1976), is currently under investigation. ACKNOWLEDGMENTS This work was supported by U.S. Public Health Grant CA-17613 and CA-12376 from the National Cancer Institute, Division of Cancer Cause and Prevention. We gratefully acknowledge the technical assistance of Mr. Joseph Rudick and the editorial assistance of Mrs. Linda Stempel, Ms. Judy Benvin, and Mrs. Nancy McNeilly. This publication is dedicated to the founder of the American Health Foundation, Dr. Ernst L. Wynder, on the occasion of the 10th anniversary of the Naylor Dana Institute for Disease Prevention.
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REFERENCES BANNASCH, P. (1976). Cytology and cytogenesis of neoplastic (hyperplastic) hepatic nodulIes. Cancer Res. 36, 2555. BARBASON, H., and BETZ, E. H. (1981). Proliferation of preneoplastic lesions after discontinuation of chronic den feeding in the development of hepatomas in rat. Brit. J. Cancer 44, 561. CASTAGNA, M., and CHAUVEAU, J. (1969). Separation des hepatocytes isoles de rat en fractions cellulaires metaboliquement distinctes. Exp. Cell Res. 57, 211. DOOLITTLE, R. L., and RICHTER, G. W. (1981). Isolation and culture of Kupffer cells and hepatocytes from single rat livers. Lab. Invest. 45, 558. DROCHMANS P., WANSON J. C., and MOSSELMANS R. (1975). Isolation and subfractionation on Ficoll gradients of adult hepatocytes. J. Cell Biol. 66, 1. EMMELOT, P., and SCHERER, E. (1980). The first relevant cell stage in rat liver carcinogenesis: A quantitative approach. B&him. Biophys. Acta 605, 247. FARBER, E. (1980). The sequential analysis of liver cancer induction. Biuchim. Biophys. Acta 605,149. GLENNER, G. G., FOLK, J. E., and MCMILLAN, P. J. (1962). Histochemical demonstration of a gamma-glutamyl transpeptidase-like activity. J. Histochem. Cytochem. 10, 481. HIROTA, N., and WILLIAMS, G. M. (1979). The sensitivity and heterogeneity of histochemical markers for altered foci involved in liver carcinogenesis. Amer. J. Pathol. 95, 317. HORSFALL, A. C., and KETTERER, B. (1976). The fractionation of isolated liver cells from normal and carcinogen treated rat. Brit. J. Cancer 33, 96. JACOBS, J. M., PRETLOW, T. P., FAUSTO, N., PITTS, A. M., and PRETLOW, T. G. (1981). Separation of two populations of cells with gamma-glutamyl transpeptidase from carcinogen-treated rat liver. J. Nat. Cancer Inst. f%(5), 967-973. KARP, J. E., GARVIN, C. C., and BURKE, P. J. (1980). Isokinetic gradient sedimentation of normal human bone marrow cells. A simple method for isolation of proliferative granulocytic and erythroid elements. J. Nat. Cancer Inst. 64, 249. KITAGAWA, T. (1976). Sequential phenotypic changes in hyperplastic areas during hepatocarcinogenesis in the rat. Cancer Res. 36, 2534. KITAGAWA, T., IMAI, F., and SATO, K. (1980). Re-elevation of gamma-glutamyl transpeptidase activity in periportal hepatocytes of rats with age. Gann 71, 362-366. LAISHES, B. A., and WILLIAMS, G. M. (1976). Conditions affecting primary cell cultures of functional adult rat hepatocytes. 1. The effect of insulin. In Vitro 12, 521-532. PITOT, H. C., BARSNESES, L., GOLDSWORTHY, T., and KITAGAWA, T. (1978). Biochemical characterization of stages of hepatocarcinogenesis after a single dose of diethylnitrosamine. Nature (London) 271, 456. PITOT, H. C., and SIRICA, A. (1980). The stages of initiation and promotion in hepatocarcinogenesis. Biochim. Biophys. Acta 605, 191. PRETLOW, T. G., II, WEIR, E. E., and ZETTERGREU, J. G. (1975). Problems connected with separation of different kinds of cells. Znf. Rev. Exp. Path&. 14, 91. PRETLOW, T. G., II, and WILLIAMS E. E. (1975). Separation of hepatocytes from suspensions of mouse liver cells using programmed gradient sedimentation in gradient of Ficoll in tissue culture medium. Anal. Biochem. 55, 114. RABES, H. M., and SZYMKOWIAK, R. (1979). Cell kinetics of hepatocytes during the preneoplastic period of diethylnitrosamine-induced liver carcinogenesis. Cancer Res. 39, 1298. SCHERER, E., and EMMELOT, P. (1976). Kinetics of induction and growth of enzyme-deficient islands involved in hepatocarcinogenesis. Cancer Res. 36, 2544. SCHULTE-HERMAN, R., OHDE, G., SCHUPPLER, J., and TIMMERMANN-TROSIENER, I. (1981). Enhanced proliferation of putative preneoplastic cells in rat liver following treatment with the tumor promoters phenobarbital, hexachlorocyclohexane, steroid compounds, and nafeopin. Cancer Res. 41, 2556. TULP, P., WELAGEN, J. J. M. N., and EMMELOT, P. (1976). Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity. Biochim. Biophys. Acta 461, 567. WALTER, H., KROB, E. J., ASCHER, G. S., and SEAMAN, G. V. F. (1973). Partition of rat liver cells in aqueous dextran-polyethylene glycol phase systems. Exp. Cell Res. 82, 15. WILLIAMS, G. M. (1976). Functional markers and growth behavior of preneoplastic hepatocytes. Cancer Res. 36, 2640.
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WILLIAMS, G. M. (1980). The pathogenesis of rat liver cancer caused by chemical carcinogenesis. Biochim. Biophys. Acta 605, 167. WILLIAMS, G. M., and GUNN, J. M. (1974). Long-term cell cultures of adult rat liver epithelial cells. Exp. Cell Res. 89, 139. WILLIAMS, G. M., BERMUDEZ, E., and SCARAMUZZINO, D. (1977). Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro 13, 809. WILLIAMS, G. M., KLAIBER, M., PARKER, S. E., and FARBER, E. (1976). Nature of early appearing carcinogen-induced liver lesions resistant to iron accumulation. J. Nat. Cancer Inst. 57, 157. WILLIAMS, G. M., and YAMAMOTO, R. S. (1972). Absence of stainable iron from preneoplastic and neoplastic lesions in rat liver with &hydroxyquinoline-induced siderosis. J. Nat. Cancer Inst. 49, 685.
AND
MOLECULAR
Isolation Accumulation Naylor
Dana
PATHOLOGY
37, 101-110
(1982)
and Enrichment of Cells Resistant to Iron from Carcinogen-Induced Rat Liver Altered
HIDEKI
MORI,’
Institute
for
Received
Disease
December
BING Mu,~
Foci
AND GARY M. WILLIAMS
Prevention, American Health Valhalla, New York IO595 21, 1981, and in revised
form
Foundation,
April
1 Dana
Road.
20, 1982
Enrichment of cells from rat liver iron-excluding foci induced by 2-acetylaminofluorene was performed using density gradients. Rats were continuously fed a diet containing the carcinogen for 8 to 13 weeks to induce altered foci that were iron excluding and positive for y-glutamyl transpeptidase activity. At weekly intervals, and after loading with iron by subcutaneous injection, the livers were perfused with collagenase and dissociated. Fractionation of dissociated control and treated liver cells on Ficoll gradients yielded three cellular fractions. In preparations from carcinogen-exposed rats the percentage of hepatocytes excluding iron and positive for y-glutamyl transpeptidase was always higher in the top (least dense) fraction than in the other lower fractions or the original population of dissociated hepatocytes. Moreover, the total number of hepatocytes in the top fraction increased progressively with the duration of feeding. These observations indicate that iron-excluding hepatocytes of enzyme-altered foci are less dense than other hepatocytes and can be enriched by gradient centrifugation. The progressive increase in the number of these altered liver cells with continued carcinogen exposure is consistent with a role for these cells in the development of liver tumors.
INTRODUCTION The altered focus is a small lesion of abnormal hepatocytes which appears in rat liver soon after exposure to experimental hepatocarcinogens and is regarded as an important precursor lesion for hepatic nodules and carcinomas (Bannasch, 1976; Barbason and Betz, 1981; Emmelot and Scherer, 1980; Farber, 1980; Kitagawa, 1976; Pitot et al., 1978; Pitot and Sirica, 1980; Rabes and Szymkowiak, 1979; Scherer and Emmelot, 1976; Schulte-Herman et al., 1981; Williams, 1976, 1980). If cells from such foci could be isolated, they would be useful for a variety of biological and biochemical studies to better define the nature of early alterations in hepatocarcinogenesis. The property of exclusion of cellular iron was developed by Williams and colleagues as a marker for abnormal populations of liver cells, including altered foci (Williams and Yamamoto, 1972; Williams, 1976, 1980). Theoretically, this property could result in a difference in the density between iron-excluding cells from altered foci and iron-containing normal hepatocytes prepared from iron-loaded livers and could thus be useful for the isolation of the altered cells on density gradients. Equilibrium density centrifugation has been applied to suspensions of liver cells to isolate hepatocytes by a number of authors (Castagna and Chauveau, 1969; ’ Present address: Department of Pathology, Gifu University School of Medicine, 40 TsukasaMachi, Gifu City 500, Japan. 2 Visiting pathologist from Shanghai Medical College of the Ministry of Railways, Shanghai, People’s Republic of China. 3 To whom correspondence and requests for reprints should be sent. 101 0014-4800/82/040101-10$02.00/O Copyright @ 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
102
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AND
WILLIAMS
Walter et al., 1973; Drochmans et al., 1975, Pretlow and Williams, 1975; Tulp et al., 1976; Horsfall and Ketterer, 1976). Among density gradient techniques, continuous gradients have been reported to be more effective than discontinuous ones (Pretlow et al., 1975; Karp et al., 1980). Pretlow and Williams (1975) also reported that velocity sedimentation was superior to isopycnic centrifugation for the separation of hepatocytes. In the present study, we attempted to isolate, by a velocity sedimentation method using continuous gradients of Ficoll, cells from iron-excluding enzymealtered foci induced in rat liver by feeding a diet containing 2-acetylaminofluorene (AAF). Following production of hepatic siderosis by iron injection, livers were dissociated by collagenase perfusion and single-cell suspension were fractionated. Iron-excluding cells from foci were found to sediment in less dense regions of the gradients than iron-containing hepatocytes and thus could be enriched. A progressive increase in the proportion of the iron-excluding population resulted from continued feeding of the carcinogen, indicating the importance of these altered cells in liver tumor development. MATERIALS
AND METHODS
Liver Cell Isolation Livers containing cells from enzyme altered foci were obtained from 18 male F344 rats (Charles River) which had been fed a diet containing 0.02% AAF for 8- 13 weeks, starting at 8 weeks of age. Four control rats were fed the basal diet NIH-07. In order to produce hepatic siderosis, rats were injected subcutaneously with 0.1 ml iron dextran (nonemic, Burns-Biotec) per 50 g body wt three times a week for 3 weeks prior to killing. Three rats were used for cell isolation every week for 6 weeks beginning after 8 weeks of carcinogen feeding. Liver dissociation was performed according to the procedures described by Williams et al. (1977). Briefly, the liver was perfused through the portal vein with 0.5 mM ethylene glycol bis(P-aminoethyl ether)-iV,N’-tetraacetic acid (EGTA; Sigma Chemical Co.) in Hanks’ Ca2+ + Mg 2+-free balanced salt solution (Flow Laboratories, Inc.) buffered with 0.01 M Hepes (Calbiochem-Behring Corp.), pH adjusted to 7.35 with 1 N NaOH for 4 min; and then perfused with 100 units/ml collagenase (Sigma, Type 1) in Williams’ Medium E (WME; Flow Laboratories, Inc. (Williams and Gunn, 1974)) buffered with 0.01 M Hepes, pH adjusted to 7.35 for 12 min. After perfusion, cells were detached by gentle combing with a stainless-steel round-tooth comb and transferred to 50-ml centrifuge tubes, pipetted, sedimented at 1OOgfor 5 min, and then resuspended in WME. This technique routinely provides a yield of 203 + 78 x lo6 cells per 100 g body wt with 79 +- 7% viability (Williams et al., 1978). The cell suspension was then incubated at 37°C with the addition of DNase (Sigma), 2.2 mg/ml phosphate-buffered saline (PBS; composition in g/liter: 8.0 NaCl, 0.20 KH2POI, 1.15 Na,HPOJ per 10 ml of cell suspension. This was followed by addition of protease (Type VI, Sigma), 5 mg/ml PBS per 10 ml of cell suspension for 15 min. After the incubation, the suspension was filtered with 25-pm Nitex. Viability was quantified by trypan blue exclusion. Density Gradients Linear density gradients of Ficoll400 (average molecular weight-400,000 Pharmacia Fine Chemicals) in PBS were constructed in 15-ml polycarbonate centrifuge
ENRICHMENT
OF ALTERED
LIVER
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103
tubes with the use of a lo-ml-capacity two-chambered gradient maker (Buchler Corp.). The gradients were constructed with the use of 5 ml of 8% (w/w) Ficoll solutions and 5 ml of 35% (w/w) Ficoll solutions on top of a I.5ml cushion of 40% Ficoll. Refractive indices of the gradients were measured with a refractometer (Bausch and Lomb) and the linearity of the gradients was confirmed. Liver cell suspensions of 1.5 x IO6 cells were layered on top of the gradients. The gradients were then centrifuged (Sorvall GLC-2B) at room temperature at 96g for 20 min using a swinging bucket rotor. Following the centrifugation, a volume equal to that of the added cells was discarded from the top of each gradient and the remainder of the gradient was collected in four fractions from the top in volumes of 4, 3, 1.5, and 1.5 ml. Hepatocytes were located mainly in the lower third of the gradients. For removal of Ficoll, each fraction was resuspended in WME and centrifuged at 800 t-pm for 5 min. After sedimentation these were resuspended and counted, together with determination of viability by trypan blue exclusion. Aliquots of cells were smeared on glass slides and air-dried. The attached cells on slides were examined microscopically after staining for iron (Prussian blue) or y-glutamyl transpeptidase (GGT) by standard methods (Hirota and Williams, 1979). The cell diameter was measured with a microreticle attached to the microscope. RESULTS Cell Isolation Perfusion of the liver with collagenase destroyed the normal sinusoidal endothelial structure and trabecular pattern of hepatocytes, as previously described (Williams et al., 1977). Nevertheless, foci of GGT-positive, iron-excluding cells could still be identified (Figs. 1 and 2). In control rats, no GGT-positive periportal hepatocytes, as seen in older animals (Kitagawa et al., 1980), were present. The
FIG. 1. Postperfusion liver from a rat fed AAF for 8 weeks and then loaded with iron. A hepatocelIular population showing positive GGT activity is located in the periportal region. GGT stain, X 144.
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FIG. 2. Serial section of the liver illustrated in Fig. 1. The GGT-positive hepatocellular population is also resistant to iron accumulation, seen as dark reaction product in surrounding hepatocytes. Iron stain, X 144.
combing procedure used to detach hepatocytes leaves behind a fibrovascular connective tissue plug containing bile ducts (Laishes and Williams, 1976) whose cells are also GGT positive (Glenner et al., 1962). After the dissociation and Nitex filtration of cells, about 80% were present as single cells and the viability by trypan blue exclusion was approximately 95% in preparations from control rats and at all stages of AAF feeding. In preparations from control rats not exposed to AAF, no GGT-positive cells were present and over 9% of cells contained stainable iron. In preparations from carcinogen-exposed rats, diffuse or granular cytochemical activity of GGT was present in cells (Fig. 3), the proportion depending upon the duration of exposure. Likewise, iron-excluding cells were identified in these preparations (Fig. 4). Cell Separation
Of the four fractions collected from Ficoll gradients, the top fraction (4 ml) contained mainly cell debris, nuclei, red blood cells, and small bile duct cells. Almost all viable cells were found in the lower three fractions. The viability of cells in these three fractions was 60-70% and was similar in each fraction from both control and exposed rats at each interval of feeding. The total recovery (total living cells from three fractions/living cells layered) was 45- 55% and was also similar at all stages. Aggregated cells were generally seen in the bottom fraction. The proportion of viable cells in each fraction and its progressive change with feeding of AAF are shown in Fig. 5. The number of cells in upper fraction 2 gradually increased with the duration of AAF feeding while that of lower fraction 4 decreased.
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FIG. 3. Isolated hepatocytes from fraction 2 from a rat given AAF for 9 weeks. Positive activity of GGT is present in several dark cells in the lower right. GGT stain, x575.
Enrichment
of Altered
Hepatocytes
The preparations from control rats yielded cell fractions that were greater than 9% iron containing and completely negative for GGT. For preparations from carcinogen-exposed rats, the percentage of altered single cells with GGT activity among the total single cells is shown in Fig. 6. The percentage of GGT-positive
FIG. 4. Isolated hepatocytes from fraction 2 from a rat given AAF for 9 weeks. Two cells in the upper left are deficient in iron accumulation. Iron stain, x575.
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FIG. 5. Proportion AAF feeding. Open
AND
WILLIAMS
OF AAF FEEDING
of viable hepatocytes in each fraction bars, fraction 2; stippled bars, fraction
and its progressive change 3; bars with oblique lines,
with stages of fraction 4.
cells in fraction 2 was always higher than in the other lower fractions (3 and 4) or in the original population of cells before the centrifugation. In particular, this separation was prominent in the early stages of AAF feeding; thus, the enrichment of GGT-positive cells in fraction 2 at 8 weeks was more than fourfold over that in fraction 4 and 3.5 times that of the original cell population. A similar enrichment occurred for iron-excluding altered cells (Fig. 7) (as iron-excluding cells, both those with complete absence of iron reaction and those with very weak reaction were counted). In general, the percentage of iron-resistant cells in each fraction was relatively higher than that of GGT-positive cells (compare Figs. 6 and 7). Characteristics
of Altered
Cells
The mean diameter of GGT-positive cells versus that of GGT-negative cells in each fraction is shown in Table I. The diameter of GGT-positive or -negative cells I4 -
. - FRACTION
WEEKS
FIG. 6. Percentage the dissociate. Mean
of GGT-positive value from three
OF MF
4
FEEDING
hepatocytes in the total rats at each week.
single
hepatocytes
of each fraction
or
ENRICHMENT
LP 0 6
OF
9
ALTERED
I II
IO WEEKS
LIVER
OF AAF
CELLS
12
107
.
I 13
FEEDING
FIG. 7. Percentage of iron-excluding hepatocytes in the total single hepatocytes of each fraction or the dissociate. Mean value from three rats at each week.
in the upper fraction was slightly less than that of the lower fractions and the diameter of GGT-positive cells increased gradually during carcinogen feeding. Generally, up to 10 weeks of AAF feeding, the mean diameter of GGT-positive cells was less than that of GGT-negative cells, but after the 10 weeks, the mean size exceeded that of negative cells, which were of the same size throughout the AAF feeding. DISCUSSION In this study, liver cells from AAF-exposed rats that were loaded with iron prior to killing were dissociated and separated on Ficoll gradients in an effort to enrich cells from iron-excluding altered foci. The total number of cells in the less dense upper fraction of the gradient increased during carcinogen feeding while that of the denser lower fractions decreased, and the percentage of iron-excluding or GGTpositive cells in the less dense top fraction was always higher than that in the lower fractions or in the initial dissociate. Size of GGT-Positive
TABLE I or -Negative Hepatocytes in Each Fraction
Diameter (pm) of GGT-positive Fraction 2 3 4
hepatocytes”
8 weeks
9 weeks
10 weeks
11 weeks
12 weeks
13 weeks
20.6 f 2.1’ 21.4 f 2.2 22.7 f 2.3
20.4 f 2.3 22.1 f 2.5 23.1 + 2.4
22.0 -c 2.5 22.8 + 2.6 24.0 -c 2.9
23.0 t 3. I 23.8 f 3.4 24.9 2 3.5
23.4 t 3.1 24.4 f 3.6 24.9 -c 3.7
23.5 + 3.5 25.1 f 3.6 26.0 k 3.2
Diameter (pm) of GGT-negative hepatocytes* 22.6 ‘- 3.0 23.9 f 3.1 24.4 f 3.2
a Mean value from three rats (100 random single cells/rat) at each week. b Mean value from total rats through 8- 13 weeks. ’ The difference in size of cells between each fraction of each interval of feeding is statistically significant (P < 0.05) except for fraction 3 compared to fraction 4 at 12 weeks. The difference in size of cells in fraction 2 at each interval of feeding is statistically significant except comparison between Week 8 and Week 9.
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The enriched iron-excluding, GGT-positive cells must be derived from the carcinogen-induced altered foci composed of cells with the same characteristics. They could not represent zonally GGT-positive cells seen in older animals (Kitagawa et al., 1980) since these were not present in the livers of the young rats used in this study and thus, no GGT-positive cells occurred in fractions from control rats. Moreover, such cells are not iron excluding, which represents one of the advantages of this property as a marker. Also, the enriched cells were too numerous and too large to be bile duct cells, which in any event are largely excluded from the cell preparations by the technique used. Thus, the present results demonstrate that iron-excluding cells from altered foci induced by AAF are relatively lighter than hepatocytes that accumulate cellular iron and can be enriched by taking advantage of their resistance to iron accumulation. The percentage of iron-excluding or GGT-positive altered cells in the upper fraction was particularly high in the early stages of carcinogen exposure. This seemed to be related to the size of the hepatocytes; i.e., the mean diameter of GGT-positive cells in the early stages was smaller than that in the later stages. Thus, separation of altered cells from hepatocytes could be most effective during the initial stages of appearance of altered foci. The percentage of iron-excluding cells in each fraction was generally greater than that of the corresponding GGT-positive cells. This may relate to the fact that the property of iron exclusion is more developed early in carcinogen administration than the enzyme histochemical abnormalities involving GGT, adenosine triphosphatase, and glucose 6-phosphatase (Hirota and Williams, 1979). Moreover, the present data show that the property of iron exclusion is also reliable as a marker for altered cells in dissociated single-cell preparations. Isolation of hepatocytes on density gradients has been reported by several laboratories (Castagna and Chauveau, 1969; Walter et al., 1973; Drochmans et al., 1975; Pretlow and Williams, 1975; Tulp et al., 1976; Horsfall and Ketterer, 1976). Recently, Doolittle and Richter (198 1) used differential centrifugations to separate Kupffer cells from hepatocytes. However, the isolation of altered cells from animals treated with hepatocarcinogens has not been extensively studied. Horsfall and Ketterer (1976) reported fractionation of isolated liver cells from rats treated with N,N-dimethyl-4-aminoazobenzene. Interestingly, they noted an increase in slowly sedimenting cells similar to the present findings, but could not identify these cells as originating in preneoplastic foci. Using GGT as a marker for altered cells, Jacobs et al. (1981) reported the enrichment of two GGT-positive populations from livers of ethionine-treated rats. One of these was suggested to represent “oval” cells and the other probably contained cells from foci. In the present study, enrichment of altered cells from foci was also obtained, but their complete purification was not achieved. Modification of the methods of density gradient isolation in combination with other methods, such as cytotoxic selection of cells from foci (Williams et al., 1976), is currently under investigation. ACKNOWLEDGMENTS This work was supported by U.S. Public Health Grant CA-17613 and CA-12376 from the National Cancer Institute, Division of Cancer Cause and Prevention. We gratefully acknowledge the technical assistance of Mr. Joseph Rudick and the editorial assistance of Mrs. Linda Stempel, Ms. Judy Benvin, and Mrs. Nancy McNeilly. This publication is dedicated to the founder of the American Health Foundation, Dr. Ernst L. Wynder, on the occasion of the 10th anniversary of the Naylor Dana Institute for Disease Prevention.
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WILLIAMS, G. M. (1980). The pathogenesis of rat liver cancer caused by chemical carcinogenesis. Biochim. Biophys. Acta 605, 167. WILLIAMS, G. M., and GUNN, J. M. (1974). Long-term cell cultures of adult rat liver epithelial cells. Exp. Cell Res. 89, 139. WILLIAMS, G. M., BERMUDEZ, E., and SCARAMUZZINO, D. (1977). Rat hepatocyte primary cell cultures. III. Improved dissociation and attachment techniques and the enhancement of survival by culture medium. In Vitro 13, 809. WILLIAMS, G. M., KLAIBER, M., PARKER, S. E., and FARBER, E. (1976). Nature of early appearing carcinogen-induced liver lesions resistant to iron accumulation. J. Nat. Cancer Inst. 57, 157. WILLIAMS, G. M., and YAMAMOTO, R. S. (1972). Absence of stainable iron from preneoplastic and neoplastic lesions in rat liver with &hydroxyquinoline-induced siderosis. J. Nat. Cancer Inst. 49, 685.