Aging lowers steady-state antioxidant enzyme and stress protein expression in primary hepatocytes.

It has been reported that the isolation and culture of primary hepatocytes can compromise cellular ability to constituitively express antioxidant enzyme (AE) genes, making it difficult to study their regulation ex vivo. In the present study, the steady-state expression of manganese-containing superoxide dismutase, copper- and zinc-containing superoxide dismutase, catalase, and glutathione peroxidase was assessed in primary hepatocytes isolated from young and senescent rats and cultured in MATRIGEL: There was no change in steady-state superoxide dismutase protein or activity levels in cells collected from young animals and cultured for 7 days. Catalase expression was initially increased, and then it declined 30%. In contrast, superoxide dismutase expression declined 60% and catalase expression declined 50% in cells from senescent animals. Constitutive and inducible 70-kDa heat shock protein expression increased coincident with declining AE levels in the young cells but not senescent cells. For both age groups, electron micrographs showed rounded hepatocytes with abundant rough endoplasmic reticulum, mitochondria, and peroxisomes. Hepatocytes were organized into clusters of 6-12 cells surrounding a large central lumen devoid of microvilli. Each cluster also contained smaller microvilli-lined lumens between adjacent hepatocytes that resembled canniculi. The plasma membranes of these lumens were sealed from the extracellular space by junctional complexes. Gap junctions in the plasma membrane suggest that hepatocytes were capable of intercellular communication. We conclude that the Matrigel system can be used to study AE regulation in primary hepatocytes from young and senescent animals, provided that experiments can be conducted within a time frame of 5-7 days in culture. These data also support the hypothesis that aging compromises hepatocellular ability to maintain AE status and upregulate stress protein expression.

Nuclear-labeling index analysis (NLIA), a software package used to perform accurate automation of cell nuclear-labeling index analysis on immunohistochemically stained rat liver samples.

The nuclear labeling index (labeled nuclei/100 nuclei) and the apoptotic index (apoptotic cells/100 cells) are important parameters of cell growth and death. Automatic counting of labeled nuclei is desirable since manual counting is tedious, time-consuming, and with a greater potential for inaccuracies. A nuclear-labeling index analysis (NLIA) software package was developed in this laboratory to perform the counting process automatically and accurately. This software package consists of an application program NLIA and a set of macros for obtaining nuclear data that is used in Scion Image. It is designed to work cooperatively with Scion Image, Adobe Photoshop, and Microsoft Office. NLIA has two basic functions: building nuclear data files and analyzing nuclear data. A color image captured from an immunohistochemically stained or autoradiographic sample is loaded into NLIA. Nuclear data can be entered into the program manually, automatically, or in combination. In the manual data entering mode, NLIA acts as an object-counting tool, while in the automatic mode it acts as a data picker: picking up the data generated by Scion Image into memory. A method to enter nuclear data (both labeled nuclei and unlabeled nuclei) in the automatic mode is described. The color image is processed in Adobe Photoshop, where the interested color ranges are selected and separated. These are then analyzed in Scion Image with the help of the macros for obtaining nuclear data. Since the advanced particle analysis function is used, the counting process is automatic and rapid. Data from thousands of nuclei can be obtained within seconds. To ensure the accuracy of the analysis, a nuclear data checking and edit feature is employed in NLIA: results of computer-generated counting can be compared with the original color image by overlaying the plot of counting results onto the original color image. In this way any computer counting mistakes can be easily discovered and corrected by the operator. Corrected nuclear data (including nuclear size, location, shape) are then stored in data files. These data files can be used in NLIA to obtain cell density and nuclear labeling indices. Because criteria for obtaining nuclear data (truncation diameter, shape factor) can be set by the operator in NLIA, nuclear size distribution and shape variation can be analyzed. This method provides a fast and accurate way to determine cell nuclear-labeling indices. Currently, Scion Image is a freeware on the internet, and NLIA software package is available from our lab home page. Methods presented here expand the Scion Image ability to analyze color images by using color separation techniques in a commercial graphic application. The instrumentation required can be relatively inexpensive, and the methods described may be useful in studies of cell kinetics, lesion growth, and tumor therapy.

The effect of amino acid composition of serum-free medium on DNA synthesis in primary hepatocyte cultures in the presence of epidermal growth factor.

The presence of optimal nutritional elements in cell culture medium is very important in studies of cultured cells. For this reason, several researchers have experimented with adding or increasing the concentration of one or more amino acids to the medium they were using to determine "essential" amino acids and optimal concentrations. We studied how leaving out one amino acid at a time from Dulbecco's modified Eagle's medium would affect epidermal growth factor-induced DNA synthesis in primary hepatocytes of the rat. Our "modified" DMEM contained only eight amino acids: arginine, cysteine, isoleucine, leucine, lysine, phenylalanine, tryptophan, and valine. Proline was found to be an essential amino acid in normal DMEM but not in the modified DMEM, and some other amino acids reduced DNA synthesis in this medium. This study showed that perhaps no single amino acid such as proline can be called "essential," but rather an optimal balance of amino acids is required for each major function of each cell type cultured.

Differences in the expression of connexin genes in rat hepatomas in vivo and in vitro.

Gap-junctional intercellular communication (GJIC) in normal rat liver cells involves at least three different connexins (Cxs)--Cx32, Cx26, Cx43--depending on the cell type, position in the lobule, or both. Whereas rat hepatocyte primary cultures expressed Cx32 and Cx26 as observed in vivo, cell lines derived from normal rat liver (WB-F344, Clone 9, RLEC, and BRL) expressed Cx43 and to a lesser extent Cx26. Hepatoma cells propagated in vitro were either deficient in GJIC and Cx expression (7777, 8994, H4IIE-C3) or communicated via gap junctions composed of Cx43 protein (N1S1-67, 9618A). Analysis of neoplasms that resulted from injection of hepatoma cells into rat femoral muscle showed differences in Cx expression when compared with cells grown in vitro. Whereas hepatoma cells 7777 and H4IIE-C3 failed to express Cx mRNAs in culture, these cells transplanted in vivo expressed levels of Cx32 mRNA comparable to those in normal liver. However, detectable Cx32 immunostaining was observed in less than 5% of the neoplastic cells in vivo. These results indicate that Cx32 protein was posttranscriptionally downregulated in 7777 and H4IIE-C3 tumor cells. Unexpectedly, 9618A cells expressed Cx43 mRNA and protein in cell culture but expressed Cx32 mRNA in vivo. In contrast, N1S1 transplants continued to express Cx43 mRNA and protein in vivo. Unlike the punctate Cx43 staining observed in suspension cultures of N1S1 cells, diffuse intracellular Cx43 staining was observed in N1S1-derived neoplasms in vivo, although the electrophoretic pattern of Cx43 isolated from N1S1 tumors grown in vivo (43 kDa) was different from that observed in suspension cell cultures (43 and 45 kDa). Thus, the findings reported here demonstrate that Cx expression in hepatoma cells depends on the environment, whether in vivo or in vitro, in which the cells are propagated.

Tamoxifen induces hepatic aneuploidy and mitotic spindle disruption after a single in vivo administration to female Sprague-Dawley rats.

Tamoxifen has found extensive use in the treatment of all stages of human breast cancer. The efficacy of tamoxifen treatment for the prevention of second primary tumors and its chemosuppressive action in animal models have led to initiation of clinical trials to test its efficacy for prevention of this disease in women. Recently, tamoxifen has been shown to induce hepatocellular carcinomas in rats. For determination of the mechanism of induction of these tumors and assessment of the possibility of risk of human cancer development from tamoxifen treatment, female Sprague-Dawley rats (five rats per treatment) were administered tamoxifen at doses ranging from 0.3 to 35 mg/kg. One day after treatment, the rats were sacrificed, and the hepatocytes were isolated and cultured for 50 h. Colcemid was added 3 h prior to harvest, and the hepatocytes were then prepared for karyotypic evaluation. One hundred metaphase spreads were examined per animal. Tamoxifen treatment resulted in the induction of aneuploidy in approximately 70% of the examined hepatocytes at the doses used. In addition, premature condensation (2-10%) and endoreduplication (5-10%) were observed in hepatocytes of rats treated with tamoxifen. Furthermore, exchanges between chromosomes as well as chromosome breakage were observed. Examination of the cultured hepatocytes from rats treated with tamoxifen by electron microscopy demonstrated both unipolar spindles and incompletely elongated spindles. Exposure of rats to a single in vivo dose of tamoxifen produced multiple changes in rat hepatocytes including clastogenic damage at doses comparable to that administered to humans. The occurrence of aneuploidy induction, premature condensation, chromosome breakage, and improper mitotic spindle formation indicates that risk versus benefit of tamoxifen treatment should be carefully evaluated.

Reestablishment of cell polarity of rat hepatocytes in primary culture.

The cell-basement membrane interaction is an important determinant of epithelial cell polarity. Although hepatocytes in situ are polarized, no morphologically identifiable basement membrane is found at their basal surface. However, several studies have demonstrated immunoreactivity to basement membrane proteins in the space of Disse, indicating the existence of an extracellular matrix, albeit of low density. Therefore we hypothesized that the interaction of hepatocytes with this matrix may determine their polarity and asked whether a basement membrane-like substrate could reestablish hepatocyte polarity in vitro. For this purpose, established monolayers of primary rat hepatocytes were cultured overlaid with a basement membrane-like matrix extracted from the Engelbreth-Holm-Swarm mouse tumor, mimicking thus the in situ tissue architecture. The hepatocytes in this culture configuration, unlike hepatocytes in classic cultures, developed distinct membrane domains, as demonstrated by the reformation of gamma-glutamyltranspeptidase, Mg(2+)-ATPase-positive bile canalicular networks and intercellular gap junctions immunolocalized to the lateral membrane with antibodies to connexin 32. The actin cytoskeleton of these cells reorganized into pericanalicular webs, and no accumulation of "stress" filaments was found beneath the membrane facing the medium. Golgi complexes appeared to be preferentially located in mitochondria-poor pericanalicular cytoplasm, indicating the polarized distribution of these organelles. Together, these data indicate that a basement membrane-like substrate present between hepatocytes and nutrient medium restores the polarity of these cells in culture. Extrapolation of these findings to the intact liver suggests that the matrix in Disse's space governs the development of hepatocyte polarity.

Effect of age on the formation of small-cell colonies in cultures of primary rat hepatocytes.

The proliferation of primary cultured rat hepatocytes was observed in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml of epidermal growth factor. These proliferating cells were mainly mononucleate and formed small-cell colonies after Day 4. The small cells in focal colonies were surrounded by typical hepatocytes and were stained immunocytochemically with anti-rat albumin and anti-cytokeratin 8 antibodies. This suggests that the cells in the small-cell colonies were derived from hepatocytes. The frequency of appearance of small-cell colonies with age was examined by the use of primary cultured hepatocytes isolated from the livers of rats between the ages of 3 wk and 90 wk. In the cells from 4- to 5-wk-old rats, about 58 colonies per 1000 attached cells appeared 144 h after plating; the number of colonies rapidly decreased to about 25 in 6- to 8-wk-old rats. In adult rats, about 17 colonies were seen, and only about five colonies were observed in rats more than 80 wk old.

Oxaloacetate induces DNA synthesis and mitosis in primary cultured rat hepatocytes in the absence of EGF.

Oxaloacetate (OA), one of the substrates in the tricarboxylic acid (TCA) cycle, was found to induce proliferation of primary cultured rat hepatocytes. During the 5 days of culture in a serum-free medium with 30 mM OA, hepatocyte DNA synthesis had been induced to the same degree as by culturing with 10 ng/ml epidermal growth factor (EGF). A 3% mitotic index was observed in OA-treated cells 60-70 hr after plating. The OA-induced DNA synthesis was inhibited by transforming growth factor-beta in a dose-dependent manner and was abolished by 10 mM hydroxyurea. OA and EGF induced DNA synthesis synergistically with > 91% of the nuclei of the cells entering S phase at 48-72 hr in culture. Among other substrates in the TCA cycle, pyruvate, lactate, alpha-ketoglutarate, succinate, fumarate, and malate at equimolar concentrations to that of OA also induced DNA synthesis. However, their activities were about half or less than that with OA. These results show that OA is a hepatocyte mitogen in vitro.

Small cell colonies appear in the primary culture of adult rat hepatocytes in the presence of nicotinamide and epidermal growth factor.

Colonies of small hepatocytes appeared after the culture of primary adult rat hepatocytes for 4 days in serum-free modified Dulbecco's modified Eagle's medium containing 10 mmol/L nicotinamide and 10 ng/ml epidermal growth factor. Each colony consisted of cells that had a single nucleus and a higher nucleus/cytoplasm ratio than surrounding hepatocytes, and immunocytochemically these cells were stained with albumin and transferrin. Ultrastructurally these cells had mitochondria, peroxisomes and desmosomes, indicating that they were derived from hepatocytes. When 6 x 10(5) cells were plated on 35-mm dishes, about 5.5 colonies/mm2 were observed. This result suggested that about 1.5% of adult rat hepatocytes has the potential for multiple replications and of forming a focal colony. These cell populations had higher proliferative activities than surrounding hepatocytes. DNA synthetic activity could not be inhibited by 2% dimethyl sulfoxide. Flow cytometric analysis showed that both 2N and 4N nuclei synthesized their DNA until day 4 but that the number of 2N nuclei rapidly increased at day 5. This result correlated with the observation of the appearance of small cell populations indicating that the cells of these focal colonies were predominantly diploid.

Ploidy and specific karyotypic changes during promotion with phenobarbital, 2,5,2',5'-tetrachlorobiphenyl, and/or 3,4,3'4'-tetrachlorobiphenyl in rat liver.

Polychlorinated biphenyls are a group of industrial chemicals that are widely distributed in the environment. Since these compounds occur as mixtures, studies of their possible interactive effects are important. In order to determine whether an interaction of 2,5,2',5'-tetrachlorobiphenyl (TCB) with 3,4,3',4'-TCB occurs during multistage hepatocarcinogenesis in vivo, like that previously observed in lymphocytes in vitro (L. M. Sargent et al., Mutat. Res., 224: 79-88, 1989), we exposed rats to a single initiating dose of diethylnitrosamine (DEN), 10 mg/kg after a 70% partial hepatectomy, and subsequently to 0.1 ppm 3,4,3',4'-TCB and/or 10 ppm 2,5,2',5'-TCB in the diet for 1 year. Administration of each of the TCBs alone after DEN initiation resulted in a low incidence of chromosomal damage in hepatocytes; but when the two were given together after DEN initiation, there was a more than additive effect on this parameter at both 7 and 12 months which was highly significant. Administration of the TCBs alone or in combination in the absence of DEN initiation also resulted in chromosomal damage, approaching that seen in livers of animals initiated with DEN when sacrificed at 12 months. In animals receiving 0.05% phenobarbital for a 12-month period after initiation with DEN, a significant degree of chromosomal breakage and fragment formation occurred both in hepatocytes expressing the ectoenzyme gamma-glutamyltranspeptidase (GGT) and in those that were GGT negative. However, the GGT-negative cells showed a significantly lower incidence of chromosomal damage than the GGT-positive hepatocytes. Exposure to phenobarbital for 7 months after DEN initiation resulted in no significant chromosomal damage in hepatocytes, whether GGT positive or GGT negative. Some degree of specificity in chromosomal alterations was seen in hepatocytes of animals initiated with DEN and promoted either with a combination of TCBs or with phenobarbital. The most frequent alterations seen were a trisomy of chromosome 1 or of its long arm and a monosomy of chromosome 3 or its short arm. Some chromosome 7 aberrations were also seen. The highest frequency of specific aberrations occurred in hepatocytes from rats that also bore hepatocellular carcinomas, suggestive of the hypothesis that genes involved in the development of hepatic carcinoma may reside in chromosome 1 and/or 3 of the rat.

Characteristics of small cell colonies developing in primary cultures of adult rat hepatocytes.

Phenotypes of the cells developing into small colonies after days of primary culture of adult rat hepatocytes in serum-free modified Dulbecco Modified Eagles' medium containing 10 mM nicotinamide and 10 ng/ml epidermal growth factor were analyzed immunocytochemically, cytochemically and ultrastructurally. Albumin, cytokeratin 8 and 18 were seen by immunocytochemical techniques in the cells of the small colonies at Day 6. Transferrin, alpha 1-antitrypsin, ceruloplasmin, and haptoglobin, proteins secreted by mature hepatocytes, were faintly stained in these cells as was alpha-fetoprotein. These proteins were secreted into the culture medium as evidenced by immunoblot analysis. gamma-Glutamyltransferase, alkaline phosphatase and glucose 6-phosphatase were not present in the cells of the small colonies as well as the surrounding hepatocytes at Day 6 of culture. In addition, ultrastructural examinations of the cells in the small colonies indicated that these cells not only had many characteristic mitochondria and desmosomes, but also a few small peroxisomes. Such cells, even after 20 days in culture were proliferating, as evidenced by the intranuclear presence of the proliferating cell nuclear antigen. The potential relation of these cells to hepatocytes which may serve as the principal reserve for replicating hepatocytes is discussed.

The bicarbonate ion is essential for efficient DNA synthesis by primary cultured rat hepatocytes.

Bicarbonate in the culture medium is essential for DNA synthesis of primary cultured rat hepatocytes stimulated by epidermal growth factor (EGF). When primary cultured hepatocytes in supplemented Leibovitz L15 medium were placed in a 100% air incubator, no increase in DNA synthesis was observed even after stimulation by EGF. However, when these cells were cultured with NaHCO3 and EGF and placed in a 5% CO2:95% air incubator, a stimulus of DNA synthesis more than 10-fold greater than in cultures in air only was seen, and many mitotic figures could be identified. Furthermore, NaHCO3 added to supplemented DMEM/F12 medium enhanced the DNA synthesis of primary cultured rat hepatocytes in this medium. The ideal pH of the medium for DNA synthesis of cultured hepatocytes was in the range of 7.6 to 8.0. A dose response of NaHCO3 in several media showed that DNA synthesis of the cells increased as the concentration of NaHCO3 increased and that 25 to 30 mM NaHCO3 in the medium was optimal for the replication of DNA by primary cultured rat hepatocytes.

Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.

Multiple cell cycles occur in rat hepatocytes cultured in the presence of nicotinamide and epidermal growth factor.

Multiple rounds of cell division were induced in primary cultures of rat hepatocytes in serum-free medium containing 10 mmol/L nicotinamide and 10 ng epidermal growth factor/ml. Cells per culture almost doubled between day 1 and day 5. The proliferating cells were predominantly mononucleate. The time course of DNA synthesis in cultured hepatocytes showed that peaks of the incorporation of 3H-thymidine were observed at 60 hr and 82 hr after plating. Labeling indices of the cells indicated that almost half the cells were labeled with 3H-thymidine in the periods 48 to 72 hr and 72 to 96 hr after plating. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 hr in culture, as demonstrated by the use of continuous treatments with 3H-thymidine and 5-bromo-2'-deoxyuridine. Furthermore, by day 4 of culture, about 40% and 15% of metaphases resulted from a second and third round of cell division, respectively. The cultured hepatocytes on day 5 stained with albumin immunocytochemically, and the activity of tyrosine aminotransferase was induced by dexamethasone and glucagon on day 3. In addition, electron micrographs revealed that dividing cells not only had many characteristics of liver mitochondria and bile canaliculus-like structures, but many also contained a few large peroxisomes with internal crystalline nucleoids.

DNA damage and repair in gamma-glutamyltranspeptidase-positive and negative hepatocytes in primary culture from carcinogen-treated rats.

Chemically induced DNA fragmentation and unscheduled DNA synthesis were determined in gamma-glutamyltranspeptidase (GGT)-positive and GGT-negative hepatocytes isolated from rat livers subjected to a multistage hepatocarcinogenesis regimen (Solt-Farber), which included 0.05% phenobarbital promotion for 6 weeks (early) or 6 months (late). The results indicated that there was DNA damage in untreated GGT-positive and GGT-negative hepatocytes with either period of promotion compared with normal hepatocytes; however, no statistical difference could be seen between GGT-positive and GGT-negative hepatocytes. DNA damage induced in vitro by the activation-dependent carcinogen dimethylnitrosamine was much less in GGT-positive hepatocytes than in GGT-negative hepatocytes or normal hepatocytes. No significant difference in DNA damage was seen in both GGT-positive and GGT-negative cell populations following treatment with the activation-independent carcinogen ethylnitrosourea (ENU), although DNA damage of GGT-positive hepatocytes was less than that of normal hepatocytes. The background of unscheduled DNA synthesis in both GGT-positive and GGT-negative hepatocytes at either time of promotion was higher than that of normal hepatocytes. The capacity for DNA repair in GGT-positive hepatocytes appeared to be lower than that in GGT-negative hepatocytes. GGT-negative hepatocytes exhibited a lower capacity for DNA repair than that of normal hepatocytes in terms of the rate of unscheduled DNA synthesis elicited by dimethylnitrosamine and ethylnitrosourea in vitro.

Quantitative stereological analysis of the effects of age and sex on multistage hepatocarcinogenesis in the rat by use of four cytochemical markers.

Altered hepatic foci (AHF) were analyzed by quantitative stereology on frozen serial sections stained sequentially for gamma-glutamyltranspeptidase (GGT), canalicular adenosine triphosphate (ATPase), glucose-6-phosphatase (G6Pase), and the placental isoenzyme of glutathione S-transferase (GST). Livers for these analyses were obtained from both male and female rats of different ages which had been subjected to initiation with a nonnecrogenic dose of diethylnitrosamine following a 70% partial hepatectomy with subsequent phenobarbital (PB) feeding. Different combinations of these four marker alterations (from single marker to four-marker combinations) were used to analyze the data, and the results were compared for their ability to detect AHF. In rats on the above protocol, GST was the single most effective marker, exhibiting a high sensitivity for scoring both number and volume of foci. There was a high degree of overlap with GGT. The combination of the four different markers, GST/GGT/ATPase/G6Pase, scored 80% more foci in number and 60% more in volume than the routinely used GGT/ATPase/G6Pase method. When all four markers were used to score AHF, PB promotion was equally effective in both sexes at weaning and at 6 months of age, but at 1 year of age males showed a dramatic reduction in the effectiveness of PB as a promoting agent, both for number and volume percentage of liver occupied by AHF. On the other hand, initiation was more effective in the male at weaning and at 6 months of age, although by the 12-month point no distinction between the sexes could be made. When only GGT was used as a marker, promotion by PB appeared to be markedly less effective in males than in females at all ages. In the absence of PB administration, both the number and volume fraction of AHF in the livers of both males and female increased with age. Likewise, both the number of AHF per liver and their volume fractions increased with age in both sexes when uninitiated animals were fed PB, although only after a 6-month lag in females. These experiments demonstrate that the stages of initiation and promotion in hepatocarcinogenesis in the rat as monitored by the number and volume percentage occupied of AHF are altered by both the age and the sex of the animal. The combination of GGT and GST identified all AHF scored by the GST/GGT/ATPase/G6Pase set of markers and thus may be the most efficient combination of markers of AHF resulting from promotion by PB.

Ploidy and karyotype of hepatocytes isolated from enzyme-altered foci in two different protocols of multistage hepatocarcinogenesis in the rat.

The ploidy and karyotypes of hepatocytes isolated from the livers of rats subjected to the protocols of Peraino et al. and of Solt and Farber were determined by the examination of such cells in primary culture. A study of 100 or more metaphases from each of five rats on each protocol revealed that 75-80% of gamma-glutamyltranspeptidase-positive (GGT+) hepatocytes isolated from livers of rats in either protocol were diploid, whereas only 23-33% of GGT- cells were diploid. Fifty percent or more of the karyotypes of hepatocytes from livers of rats receiving the Solt-Farber protocol exhibited one or more chromosomal breaks, whereas hepatocytes from livers of rats subjected to the Peraino protocol showed no increase in chromosomal breakage over that in normal controls. These studies demonstrate that the majority of GGT+ cells from altered hepatic foci are diploid and that the greater toxicity of the Solt-Farber protocol over that of Peraino is correlated with marked chromosomal breakage of GGT+ hepatocytes.

Electron microscopic and time lapse studies of mitosis in cultured rat hepatocytes.

Primary cultures of adult rat hepatocytes in a serum-free medium were observed by time lapse cinematography to proceed through mitotis and cytokinesis. An ultrastructural study of these cultures is presented with electron micrographs of each stage of mitosis and cytokinesis. The cultured hepatocytes begin to enter prophase about 48 hr after plating and proceed through mitosis in approximately 70 min not including cytokinesis. During this time, they remain somewhat flattened and joined to neighboring cells rather than rounding up. Both mononucleate and binucleate hepatocytes proceed through mitosis. Some mononucleate cells do not undergo cytokinesis, resulting in the formation of binucleate cells. In binucleate hepatocytes, both nuclei proceed through prophase simultaneously. Usually a single mitotic spindle with a large metaphase plate containing chromosomes from both nuclei is observed. Cytokinesis frequently occurs in binucleate hepatocytes which have a single mitotic spindle. Some binucleate cells form tripolar or 4-polar metaphase plates. In tripolar metaphases, some cells do not divide, resulting in multinucleate cells, whereas others undergo cytokinesis yielding three mononucleate cells or one binucleate and one mononucleate cell. Two mitotic spindles located perpendicularly to each other with microtubules intertwining have been observed in 4-polar metaphases. In this latter case, no cytokinesis has been observed. This study shows that both mononucleate and binucleate adult rat hepatocytes cultured in a serum-free medium in the presence of epidermal growth factor not only synthesize DNA, but progress through mitosis and often cytokinesis.

A method for the comparative study of replicative DNA synthesis in GGT-positive and GGT-negative hepatocytes in primary culture isolated from carcinogen-treated rats.

The presence of gamma-glutamyl transpeptidase (GGT) in focal nodules of hepatocytes is a commonly used marker for the identification of preneoplastic cell populations. Female Fischer 344 rats were initiated with a single intragastric administration of 200 mg diethylnitrosamine/kg, altered cells were selected after 0.02% 2-acetylaminofluorene was given in the diet; this was followed by a partial hepatectomy and promotion with dietary sodium phenobarbital for 4 wk. A mixed-cell population of GGT-positive and GGT-negative hepatocytes was obtained after collagenase perfusion and Percoll purification. An enriched population of GGT-positive hepatocytes was obtained by a modified "panning" technique. With quantitative scintillation spectrometry and autoradiography of [3H]thymidine incorporation, replicative DNA synthesis of GGT-positive and GGT-negative rat hepatocytes was observed in both the mixed-cell population and the enriched GGT-positive and GGT-negative cell populations. Under the culture conditions used, GGT-positive cells showed a higher level of replicative DNA synthesis than did GGT-negative cells; this indicates that such altered hepatocytes in the stage of promotion possess an inherently greater capacity for all replication, as previously suggested from studies in vivo.