The kinetics of the cell population of human liver parenchyma at different periods of life]

Processes of polyploidization in the liver parenchyma were investigated in the course of postnatal organism growth, stabilization of growth and ageing, using cytophotometry on the slides of isolated hepatocytes from normal livers of 140 donors aged from 1 day to 92 years. In addition, livers of human embryos (4, 5, 6 and 7 month old) were investigated. It is concluded that polyploid cells in the human liver appear in individuals aged from 1 to 5 years. However, during the postnatal development their relative number increases insignificantly. At the end of the intensive postnatal growth period the share of polyploid human liver cells is less than 3%. Binuclear cells with diploid nuclei are seen as early as in the embryonic liver. After birth their number increases slowly to reach 7.1% in the 16-20 year age group. The postnatal growth of human liver is due mainly to mitotic divisions of mononuclear diploid hepatocytes whose relative number is more than 90% during the postnatal growth. During the period of maturity (from 21 to 50 years), when the liver practically stops to grow, the levels of hepatocyte ploidy are changed insignificantly: part of 2c-hepatocytes decreases slowly (up to 84.8% by the end of period) and (2c x 2)-hepatocyte number increases slowly too. The number of polyploid cells increases by several times, but is equal only to 6.6% of all the hepatocytes counted. Under ageing, on the background of human liver atrophy, acceleration of hepatocyte polyploidization takes place. In the age group of 86-92 years parts of 2c- and (2c x 2)-hepatocytes reach 60.3 and 14.3%, resp., and the total share of polyploid cells is as much as near 25%, calculated from the cell population of liver parenchyma. The maximum ploidy levels in hepatocytes of normal human liver during ageing is becoming 16c and 8c x 2 for mononuclear and binuclear cells, resp. Transition rates among hepatocytes of different ploidy classes (2c--2c, 2c--2c x 2, 2c x 2--4c, 2c--4c) were calculated in addition to the coefficient of changing of the hepatocyte proliferative activity with the increase in its ploidy and cell death rate in different periods of human life. A rather high hepatocyte proliferative activity in the early postnatal period of human life was seen to lower during the following years of life. In maturity it is the lowermost to make less than 5% of that in newborns. During ageing the hepatocyte DNA-synthesizing activity being almost 1.6-1.7 times as much as in maturity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polyploidizing mitoses and the biological meaning of polyploidy in liver cells]

The ontogenetic polyploidization of hepatocytes is regarded, within which normal mitoses are changed to polyploidizing mitoses, and diploid hepatocytes transform into polyploid mono- and binuclear cells. A new hypothesis is put forward of the biological significance of the liver cell polyploidy. The hypothesis takes into account a high level of spontaneous chromosomal aberrations in mitotic hepatocytes. The chromosome structural changes interfere with mitosis resulting in the chromosomal imbalance. Polyploidy bestows for hepatocytes a tolerance towards a chromosomal imbalance. Some implications of the hypothesis are discussed: unbalanced genome of hepatocytes after the treatment with mutagens and mitotic stimulators; the reasons of liver cell polyploidy differences in mammalian species; mechanisms of radioresistance of hepatocytes. Chromosomal imbalance of polyploid hepatocytes is assumed to be the basis for wome chronic liver diseases in man.     

Early effects in chemical-induced rat liver carcinogenesis: an immunohistochemical study following exposure to 0.04% AAF

To investigate effects that distinguish AAF from incomplete carcinogens, the rate of cell death (apoptosis) and cell proliferation was studied at early stages of AAF induced rat liver carcinogenesis. Male Wistar rats were fed 0.04% AAF in the diet for 2, 6 and 16 weeks and immunohistochemical markers were measured in the liver. The formation of initiated cells and preneoplastic foci was followed by staining for GST-P (glutathione-S-transferase). GST-P-positive foci were present from 6 weeks on. Apoptosis was increased in the periportal area and in preneoplastic foci at all time points. Cell proliferation was enhanced in the periportal area in oval cells and in bile duct-like cells particularly at 2 and 6 weeks and mainly in GST-P positive foci at 16 weeks. Notably, more cells always proliferated than were eliminated. Other apoptosis-related markers like p53 and FAS/Apo-1 could not be demonstrated in either normal hepatocytes, preneoplastic foci or in hepatocytes from treated animals. Scattered bcl-2 positive cells were present in livers at 16 weeks of treatment. The two cell growth and differentiation related proto-oncogenes c-FOS and c-JUN were increased in all treated animals at early stages. If feeding was stopped after 6 weeks, livers did not recover significantly within the following 10 weeks. The results support the complex effects of AAF in rat liver carcinogenesis. Chronic toxicity locally impairs the balance between cell proliferation and cell death and induces morphological alterations that promote the growth of initiated cells.     

Reduced autophagic activity, improved protein balance and enhanced in vitro survival of hepatocytes isolated from carcinogen-treated rats

Sequential carcinogen treatment (diethylnitrosamine/partial hepatectomy followed by 2-acetylaminofluorene (2-AAF] induced multiple hepatocarcinomas in rats with 100% certainty within a year. Enzyme-altered lesions, i.e. gamma-glutamyltranspeptidase (GGT)-positive and/or ATPase-negative cell foci, were numerous already at 8 weeks, and suspensions of purified hepatocytes isolated (by collagenase perfusion) at this time contained 30-40% GGT-positive cells. These hepatocyte suspensions were markedly deficient with respect to autophagic protein degradation (in comparison with cell suspensions from normal rats), and the cells lost less protein and survived much better than normal hepatocytes in culture under conditions of amino acid deprivation (which activates the autophagic mechanism). The anabolic advantage of reduced autophagy may possibly contribute to the selective outgrowth of preneoplastic cells during the earliest stage of liver carcinogenesis. Inclusion of the autophagy inhibitor 3-methyladenine in the culture medium elevated the survival of normal hepatocytes up to the level seen with hepatocytes from carcinogen-treated animals, suggesting that protection of normal cells by autophagy suppression may be a potentially interesting therapeutic principle.     

Binucleation and polyploidization patterns in developmental and regenerative rat liver growth

The hepatocellular binucleation rate, measured as the percentage of binuclear cells amongst newly formed bromodeoxyuridine-labelled and immunostained collage-nase-isolated rat hepatocytes, decreased from 12% to 4% between days 30 and 40 after birth, rose to 20% between days 50 and 60, and then declined again to the adult rate of about 10% at day 80. During regenerative growth following a two-thirds partial hepatectomy, the rate of binucleation declined to about 3%, causing the fraction of binuclear cells to fall from 27% (before hepactectomy) to 5% (at 45 h after hepactectomy) as pre-existing binuclear cells replicated and formed mononuclear daughter cells. Essentially all (97%) hepatocytes replicated at least once, starting their DNA synthesis at around 13 h and reaching a peak at 30 h, irrespective of ploidy and nuclearity. At later time points, the diploid hepatocytes had a higher labelling index than the polyploid cells, suggesting a greater tendency to go through several cell cycles.     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Intoxication of rats with CCl4 (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS+) hepatocytes were reestablished. However, while the GS+ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS+ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS+ and GS- hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS+ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS- hepatocytes. Immediately after liver damage by CCl4 and during regeneration small but significant changes in the nuclear pattern were noted for GS- hepatocytes. However, the first GS+ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS- hepatocytes. These results indicate that new GS+ hepatocytes may be derived from formerly GS- cells which are induced to express GS if they have reached the border of the central veins.     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.     

Biological significance of liver cell polyploidy: an hypothesis

The liver cell polyploidy phenomenon, a characteristic of many species of mammals, is reviewed. The liver parenchyma of adult animals represents a mixed population of mononuclear and binuclear cells with different number of chromosome sets and, therefore DNA content per nucleus. The polyploid hepatocytes are formed during postnatal liver growth as a result of a change from normal mitoses to polyploidizing ones. Hence, the polyploidization of hepatocytes is regarded as an equivalent of cell multiplication.An hypothesis of the biological significance of liver cell polyploidy is based on the fact of a high level of spontaneous chromosome aberrations in mitotic hepatocytes. Ploidy increase is known to give resistance against different kinds of genome alteration. Polyploidization of the liver cells ensures protection against deleterious consequences of the aberrant genome formation resulting from aberrant mitoses.Some implications of the hypothesis are discussed: the reasons for species-specific differences of liver cell polyploidy; the mechanisms of hepatocyte radioresistance; the relation of polyploidy to liver cell aging. The prerequisite factors for unbalanced cell genome formation are adduced: DNA and chromosome damage as the first step in the process, stimulation of mitosis as the second one. The aberrant polyploid genome of hepatocytes is assumed to be the cytogenetic basis for some chronic liver diseases in man.     

Aneuploidy,polyploidy and ploidy reversal in the liver

Polyploidy has been described in the liver for over 100 years. The frequency of polyploid hepatocytes varies by age and species, but up to 90% of mouse hepatocytes and approximately 50% of human hepatocytes are polyploid. In addition to alterations in the entire complement of chromosomes, variations in chromosome copy number have been recently described. Aneuploidy in the liver is pervasive, affecting 60% of hepatocytes in mice and 30–90% of hepatocytes in humans. Polyploidy and aneuploidy in the liver are closely linked, and the ploidy conveyor model describes this relationship. Diploid hepatocytes undergo failed cytokinesis to generate polyploid cells. Proliferating polyploid hepatocytes, which form multipolar spindles during cell division, generate reduced ploidy progeny (e.g., diploid hepatocytes from tetraploids or octaploids) and/or aneuploid daughters. New evidence suggests that random hepatic aneuploidy can promote adaptation to liver injury. For instance, in response to chronic liver damage, subsets of aneuploid hepatocytes that are differentially resistant to the injury remain healthy, regenerate the liver and restore function. Future work is required to elucidate the mechanisms regulating dynamic chromosome changes in the liver and to understand how these processes impact normal and abnormal liver function.     

The growth of preneoplastic hepatocytes in the spleen of recipient mice]

Hepatocytes and the fraction of non parenchymal cells enriched with oval cells were extracted from the preneoplastic mouse liver at the stage of hyperplastic node formation and implanted into the spleen. In 14-16 months after the transplantation, multiple islets of hepatocytes which replaced up to 25% of the spleen cut area, were found in 57% (4 of 7) and 22% (8 of 36) of recipients respectively. The hepatocytes formed 2-3-cell bulks or solid masses organized into multicellular trabecules, and expressed biliary capillary antigen, albumin and transferrin. In the inoculation of nonparenchymal cell fraction, the growth of hepatic tissue in the spleen depended on the magnitude of hepatocyte admixture to be undetectable in absence of hepatocytes in the donor suspension. The growth of hepatic tissue in spleen was observed following the injection of a small number (3 x 10(-4)-6 x 10(-5)) of live hepatocytes. This fact evidences an extremely high clonogenic potency of clonogenic potency of preneoplastic hepatocytes.     

Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Summary Intoxication of rats with CCl₄ (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS⁺) hepatocytes were reestablished. However, while the GS⁺ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS⁺ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS⁺ and GS^– hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS⁺ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS^– hepatocytes. Immediately after liver damage by CCl₄ and during regeneration small but significant changes in the nuclear pattern were noted for GS^– hepatocytes. However, the first GS⁺ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS^– hepatocytes. These results indicate that new GS⁺ hepatocytes may be derived from formerly GS^– cells which are induced to express GS if they have reached the border of the central veins.     

Liver cell polyploidization: a pivotal role for binuclear hepatocytes

Polyploidy is a general physiological process indicative of terminal differentiation. During liver growth, this process generates the appearance of tetraploid (4n) and octoploid (8n) hepatocytes with one or two nuclei. The onset of polyploidy in the liver has been recognized for quite some time; however, the cellular mechanisms that govern it remain unknown. In this report, we observed the sequential appearance during liver growth of binuclear diploid (2 x 2n) and mononuclear 4n hepatocytes from a diploid hepatocyte population. To identify the cell cycle modifications involved in hepatocyte polyploidization, mitosis was then monitored in primary cultures of rat hepatocytes. Twenty percent of mononuclear 2n hepatocytes failed to undergo cytokinesis with no observable contractile movement of the ring. This process led to the formation of binuclear 2 x 2n hepatocytes. This tetraploid condition following cleavage failure did not activate the p53-dependent checkpoint in G1. In fact, binuclear hepatocytes were able to proceed through S phase, and the formation of a bipolar spindle during mitosis constituted the key step leading to the genesis of two mononuclear 4n hepatocytes. Finally, we studied the duplication and clustering of centrosomes in the binuclear hepatocyte. These cells exhibited two centrosomes in G1 that were duplicated during S phase and then clustered by pairs at opposite poles of the cell during metaphase. This event led only to mononuclear 4n progeny and maintained the tetraploidy status of hepatocytes.     

Fractionation of Rat Hepatocyte Subpopulations with Varying Metabolic Potential, Proliferative Capacity, and Retroviral Gene Transfer Efficiency

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated thatsmalldiploid andlargepolyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.     

Adaptive responses of rat liver to the gestagen and anti-androgen cyproterone acetate and other inducers. III. Cytological changes

Treatment of rats with cyproterone acetate (CPA) induces liver growth. Intact hepatocytes and cell nuclei were isolated from enlarged livers and their volumes or diameters were determined by electronic and microscopic methods. No changes in mean hepatocyte volume or ploidy were observed. However, there was a marked fall in the frequency of binuclear hepatocytes (from 43% to 7%) and a concomitant increase of nuclear ploidy. This effect probably resulted from CPA-induced replication of binuclear hepatocytes. The total number of hepatocytes replicating in response to CPA was estimated on the basis of these data and was found to be up to 75% of all parenchymal cells. Similar cytological changes were observed in the liver after treatment with pregnenolone-16 alpha-carbonitrile (PCN) and, to a lesser extent, with alpha-hexachlorocyclohexane (alpha-HCH). In contrast, physiological liver growth in adolescent rats was characterized by only small changes in binuclearity and nuclear ploidy, and by increases of cellular ploidy. Thus, ploidy analyses may be a useful tool to characterize the type of growth stimulation. Following discontinuation of treatment the cytological changes induced by CPA or alpha-HCH were not reversible in a matter of 3 weeks.     

Growth patterns of rat hepatocytes during postnatal development.

During postnatal growth in the liver of the rat, a characteristic shift towards binuclear cells and cells of higher ploidy class occurs. When the protein content of individual isolated hepatocytes of different ploidy classes is analysed cytophotometrically using the specific protein stain Naphthol Yellow S, it appears that the growth in mass in the period 30-99 days is due mainly to increase of protein content of binuclear diploid (BD) and mononuclear tetraploid (MT) cells. The mononuclear diploid (MD) cells play a quickly diminishing role in the parenchymal population after the initial growth phase and cells of highest ploidy degree remain unimportant quantitatively. The quickly growing BD and MT cells only reach a Naphthol Yellow S protein value twice that of MD cells after a certain period of growth, whereas changes in protein content are slight or absent from 99 days onwards in all cell types investigated.     

Apoptosis in the liver and its role in hepatocarcinogenesis

Apoptosis seems to be the predominant type of active cell death in the liver (type I), while in other tissues cells may die via biochemically and morphologically different pathways (type II, type III). Active cell death is under the control of growth factors and death signals. In the liver, endogenous factors, such as transforming growth factor 1 (TGF-1), activin A, CD95 ligand, and tumor necrosis factor (TNF) may be involved in induction of apoptosis. Release and action of these death factors seems to be triggered by exogenous signals such as withdrawal of hepato-mitogens, food restriction, etc.During stages of hepatocarcinogenesis, not only DNA synthesis but also apoptosis gradually increase from normal to preneoplastic to adenoma and carcinoma tissue. Also, in human carcinomas, birth and death rates of cells are several times higher than in surrounding liver. (Pre)neoplastic liver cells are more susceptible than normal hepatocytes to stimulation of cell replication and of cell death. Consequently, tumor promoters may act as survival factors, i.e., inhibit apoptosis preferentially in preneoplastic and even in malignant liver cells, thereby stimulating selective growth of (pre)neoplastic lesions. On the other hand, regimens favoring apoptosis and lowering cell replication may result in selective elimination of (pre)neoplastic cell clones from the liver. Finally, we have studied the first stage of carcinogenesis, namely the appearance of putatively initiated cells after a single dose of the genotoxic carcinogen N-nitrosomorpholine (NNM). Most of these cells were found to be eliminated by apoptosis, suggesting that initiation, at the organ level, can be reversed at least partially by preferential elimination of initiated cells. These events may be regulated by autocrine or paracrine actions of survival factors.     

Isolation and characterization of parenchymal cells from normal and cirrhotic rat liver

A technique is described for isolation of adult rat hepatocytes from micronodular cirrhotic livers based on a collagenase digestion procedure. Hepatocytes from normal livers and those chronically injured by thioacetamide did not differ with respect to the viability measured by the trypan blue exclusion test or to the cellular concentrations of protein and glycogen, but the triglyceride content of cells from cirrhotic livers was significantly reduced. Hepatocytes isolated from cirrhotic livers are ultrastructurally in a good state of preservation but they appear to be poorer than controls in RER membranes, although the well-preserved mitochondria are somewhat richer in cristae. No differences were detected between the cell preparations in rates of gluconeogenesis and total de novo fatty acid synthesis, but the secretion of newly synthesized fatty acids was significantly reduced in cells from cirrhotic livers. Thus adult rat hepatocytes can be isolated from thioacetamide-induced micronodular cirrhotic livers with high yield and morphological integrity. Differentiated functions are maintained in suspension for at least 4 h.