Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers

An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.     

Extended primary culture of human hepatocytes in a collagen gel sandwich system

Summary To develop a strategy for extended primary culture of human hepatocytes, we placed human hepatocytes between two layers of collagen gel, called a “collagen gel sandwich.” Maintenance of hepatocellular functions in this system was compared with that of identical hepatocyte preparations cultured on dry-collagen coated dishes or co-cultured with rat liver epithelial cells. Human hepatocytes in a collagen gel sandwich (five separate cultures) survived for more than 4 wk, with the longest period of culture being 78 d. They maintained polygonal morphology with bile canaliculuslike structures and high levels of albumin secretion throughout the period of culture. In contrast, hepatocytes on dry-collagen became feature-less, and albumin secretion could not be detected after 14 d of culture. This loss of albumin secretion was partially recovered by overlaying one layer of collagen gel. Ethoxyresorufin O-deethylase activity, associated with cytochrome P450 1A2, was detected basally up to 29 d in collagen gel sandwich culture. These activities were induced four- to eightfold after induction with dibenz(a,h)anthracene. Cocultures also maintained basal activity up to 29 d. However, their inducibility was lower than that of hepatocytes in collagen gel sandwich. No ethoxyresorufin O-deethylase activity was detected in hepatocytes cultured on dry-collagen at 7 d. Thus, the collagen gel sandwich system preserves differentiated morphology and functions of human hepatocytes in primary culture for a prolonged period of time. This system is a promising model for studying human hepatocellular function, including protein synthesis and drug metabolism in vitro.     

Influence of collagen gel substrata on certain biochemical activities of hepatocytes in primary culture

In order to study the influence of cell shape as modulated by the extracellular matrix on the cellular activity, hepatocytes isolated from liver were maintained on collagen I coated plastic substrata and collage I gel substrata and certain hepatocyte specific functions were investigated. The incorporation of³[H]-leucine into total proteins and albumin secreted by cells maintained on collagen gel was found to be significantly higher compared to those maintained on a collagen coated plastic substrata, indicating that hepatocytes on collagen gel have an enhanced albumin synthesizing capacity. Increased incorporation of³⁵[S]-sulphate into total proteoglycans (PG) and a relatively higher fraction of the³⁵[S]-PG in the extracellular space showed an increased rate of synthesis and secretion of sulphated PGs by cells maintained on collagen gels. But in contrast to the above results, the incorporation of³[H]-leucine into cytokeratins C₈, C₁₈ and actin were significantly low in cells maintained on collagen gel. The tyrosine amino transferase activity exhibited by hepatocytes preincubated with dexamethasone on collagen gel was also significantly low. The different forms of collagen substrata appeared to have no effect on the amino acid transport by hepatocytes, further suggesting that the various hepatocyte specific functions are not uniformly altered when hepatocytes are maintained on three-dimensional collagen gel substrata. These results indicate that the shape of the cell as determined by the nature of the matrix substratum influences the synthetic activity of secretory proteins and those remaining intracellularly, differently.     

Interaction between collagen and adenosine 5'-triphosphate.

A quantitative study was made of the adsorption of adenosine 5′-triphosphate (ATP) on collagen by following the change in the absorbance at 258 nm of ATP in the soaking solution. The amount of ATP adsorbed decreased exponentially with the increase of pH up to pH 8 and fell off more rapidly at higher pH values. At a given pH, when the concentration of ATP was increased, the amount of ATP adsorbed increased following the pattern of a Langmuir isotherm. The adsorption was independent of the cation present. The adsorption of adenosine 5′-diphosphate was essentially the same as that for ATP. For tendons deposited with calcium phosphate, the amount of ATP adsorbed decreased compared to natural tendons. The adsorption of ATP on collagen fibers inhibited the contraction caused by calcium chloride, calcium bromide, and lithium bromide. In solution, ATP had very little effect on the denaturation of acid-soluble collagen caused by calcium chloride.     

Interaction between satellite cells and skeletal muscle fibers

Single myofibers with attached satellite cells isolated from adult rats were used to study the influence of the mature myofiber on the proliferation of satellite cells. The satellite cells remain quiescent when cultured in serum containing medium but proliferate when exposed to mitogen from an extract of crushed adult muscle. The response of satellite cells to mitogen was measured under three situations with respect to cell contact: (1) in contact with a viable myofiber and its basal lamina, (2) detached from the myofiber by centrifugal force and deposited on the substratum and (3) beneath the basal lamina of a Marcaine killed myofiber. The results show that satellite cells in contact with the plasmalemma of a viable myofiber have reduced mitogenic response. Since inhibiting growth may induce differentiation, I tested whether satellite cells proliferating on the surface of a myofiber would fuse. Although the satellite cell progeny were fusion competent, they did not fuse with the myofiber. To determine whether fusion competence of the myofiber changes with time in culture, embryonic myoblasts were challenged to fuse with myofibers that had been stripped of satellite cells and cultured for several days. The myoblasts fused with pseudopodial sprouts growing from the ends of the myofiber, but did not fuse with the original myofiber surface. These results indicate that contact with the surface of a mature myofiber suppresses proliferation of myogenic cells but the cells do not fuse with the myofiber.     

Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel.

During freezing, intracellular ice formation (IIF) has been correlated with loss in viability for a wide variety of biological systems. Hence, determination of IIF characteristics is essential in the development of an efficient methodology for cryopreservation. In this study, IIF characteristics of hepatocytes cultured in a collagen matrix were determined using cryomicroscopy. Four factors influenced the IIF behavior of the hepatocytes in the matrix: cooling rate, final cooling temperature, concentration of Me2SO, and time in culture prior to freezing. The maximum cumulative fraction of cells with IIF increased with increasing cooling rate. For cultured cells frozen in Dulbecco's modified Eagle's medium (DMEM), the cooling rate for which 50% of the cells formed ice (B50) was 70 degrees C/min for cells frozen after 1 day in culture and decreased to 15 degrees C/min for cells frozen after 7 days in culture. When cells were frozen in a 0.5 M Me2SO + DMEM solution, the value of B50 decreased from 70 to 50 degrees C/min for cells in culture for 1 day and from 15 to 10 degrees C/min for cells in culture for 7 days. The value of the average temperature for IIF (TIIF) for cultured cells was only slightly depressed by the addition of Me2SO when compared to the IIF behavior of other cell types. The results of this study indicate that the presence of the collagen matrix alters significantly the IIF characteristics of hepatocytes. Thus freezing studies using hepatocytes in suspension are not useful in predicting the freezing behavior of hepatocytes cultured in a collagen matrix. Furthermore, the weak effect of Me2SO on IIF characteristics implies that lower concentrations of Me2SO (0.5 M) may be just as effective in preserving viability. Finally, the value of B50 measured in this study indicates that cooling rates nearly an order of magnitude faster than those previously investigated could be used for cryopreservation of the hepatocytes in a collagen gel.     

Interaction between aflatoxin B1 and oxytetracycline in isolated rat hepatocytes

Isolated rat hepatocytes were used as an in vitro model to investigate A possible interaction between oxytetracycline (OXT) and aflatoxin B₁ (AFB₁). LDH leakage, RNA and protein synthesis and glycogen accumulation were measured in the presence of both drugs, either separately or in combination. The evolution of LDH leakage during the incubation was identical in untreated and treated cells. AFB₁ inhibited RNA and protein synthesis at a concentration of 10^–7 M and 10^–6 M, respectively, and higher, whereas OXT did not influence RNA synthesis but inhibited protein synthesis at the highest tested concentration, 10^–3 M. As far as glycogen is concerned, rats were injected with glucagon before sacrifice in order to obtain a constant synthesis rate in isolated hepatocytes. AFB₁ inhibited the accumulation of glycogen from 10^–6 M upward. This effect was never observed before 90 min of incubation. OXT had no effect on glycogen synthesis. In the presence of both drugs, no interaction was demonstrated as far as RNA and protein synthesis were concerned, but OXT opposed the inhibition induced by AFB₁ on glycogen accumulation. If the in vivo protection, provided by OXT against AFBI-induced toxicity, is due to a direct interference in the toxic mechanisms of the mycotoxin, these results show that OXT does not influence the AFB₁-inhibition of RNA and protein synthesis. The latter are early and sensitive parameters inhibited by AFB₁. On the contrary, taking into consideration the results on glycogen accumulation, it seems more interesting to investigate further this metabolism.Abbreviations AFB₁ Aflatoxin B₁ - OXT Oxytetracycline - DMEM Dulbecco's Modified Eagle's Medium - LDH Lactate Dehydrogenase - DMSO Dimethyl Sulfoxide - BSA Bovine Serum Albumin     

Reaction between the mechanocytes of hematopoietic organs in collagen gel

Interactions between stromal mechanocytes of hemopoietic organs were studied at their cultivation in three-dimentional collagen gel. It was demonstrated that cellular cords appearing between fibroblast colonies and between fragments of hemopoietic organs are of fibroblastic nature. They are not resulted from organic or specific peculiarities, or from discharge of substances attracting fibroblasts. A linear dependence between the amount of fibrorow or spleen was noted. Fibroblast colonies formed by the hamster bone marrow and splenic cells, as well as by passaged fibroblasts of the guinea pig bone marrow were obtained. In order to form colonies by the passaged fibroblasts it is necessary to add of irradiated cells. Its effect, besides the medium conditioning, is evidently, in restriction of fibroblast mobility in collagen gel.     

Polyelectrolyte complexes. 2. Interaction between collagen and polyanions

The electrostatic interactions that occur in connective tissue between polyanions and proteins have been studied in model systems by a technique involving a fluorescent probe, acridine orange. It was found that collagen bound more strongly than bovine serum albumin to the polyanions studied. At pH 3.0, collagen formed strong complexes of definite stoichiometry with chondroitin-4-sulphate, chondroitin-6-sulphate, heparin and polystyrene sulphonate that were stable in sodium chloride solution of 0.1 M. The complexes of collagen with hyaluronic acid, or carboxymethylcellulose were less stable. The effect of pH variations (3.0–9.0) on the binding was investigated. Critical electrolyte concentrations (NaCl) were determined for complexes of collagen with glycosaminoglycans that dissociated at salt concentrations below that at which collagen precipitates. The values obtained were, 0.1 M for hyaluronic acid, and 0.5 M for chondroitin sulphate.     

用凝胶电泳发现肝素与染料之间的相互作用

目的:观察肝素与23种染料之间的相互作用.方法:利用淀粉琼脂糖凝胶电泳及聚丙烯酰胺凝胶电泳,观察染料加肝素后电泳行为的变化.结果:淀粉琼脂糖凝胶电泳中染料加肝素后多数结果是泳速变慢,有一些留在原点,也有前移、后退者.二氯荧光素、荧光红、荧光素、甲酚红、苯胺蓝、溴酚蓝、茜素红S、胭脂红、亮绿、氨基黑10B、丽春红G、曙红、苯胺黑、氯酚红加肝素后泳速变慢.亚甲蓝、灿烂甲酚蓝、甲基紫加肝素后电泳留在原点.刚果红、洋红加肝素后电泳变化不明显.四氯荧光素、氯化硝基四氮唑蓝加肝素后电泳荧光看不清.聚丙烯酰胺凝胶电泳中溴酚蓝加入肝素后泳速明显变慢,随着肝素量增加溴酚兰的泳速加快.与淀粉琼脂糖凝胶电泳结果一致,但它的电泳结果更明显.结论:本次所研究的23种染料,几乎都能与肝素发生相互作用,给它们的进一步深入研究打下基础.     

Microfilament accumulation and the transport of amino acids and glucose in adult rat hepatocytes cultured on collagen gel/nylon mesh

Transport of α-aminoisobutyric acid in cultured hepatocytes is temperature- and energy-dependent, whereas transport of 2-deoxy-D-glucose is not energy-dependent. In early cultures of hepatocytes (day 2) on a collagen gel/nylon mesh, the cells contain few microfilaments and the transport of amino acids and glucose is 5–7 times more than in late cultures of hepatocytes (day 6) which contain an apical, extensive accumulation of microfilaments. Cytochalasin D has little effect on the transport of amino acids and glucose in day 2 cultures of hepatocytes, but enhances transport of both compounds in day 6 cultures. These findings suggest that the microfilament accumulation in cultured hepatocytes inhibits transport of amino acids and glucose.     

Interaction between cells and elastin fibers: An ultrastructural and immunocytochemical study

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting “pseudopodia” that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types. © 1994 Wiley-Liss, Inc.     

Adhesion of rat hepatocytes to collagen

Rat hepatocytes, freshly isolated with a collagenase perfusion technique, were found to attach within 1 h on collagen substrates and on culture dishes coated with cold insoluble globulin (CIG) or asialoceruloplasmin (AC). Spreading was observed on collagen and CIG but not on AC. Both attachment and spreading occurred in a simple balanced salt solution in the absence of serum. In the absence of serum no attachment was observed on plain plastic dishes or on dishes coated with serum albumin or other plasma proteins, unless divalent manganese ions were present. In the presence of manganese the hepatocytes attached to all surfaces tested, but no spreading occurred. Attachment to collagen occurred equally well to collagens type I or type III both in the native, fibrillar state and in the denatured state. Collagen attachment required magnesium ions but did not appear to involve the collagen-linked carbohydrates. Different mechanisms were found to operate in hepatocyte attachment to collagen and to AC; the latter is most likely mediated by the hepatocyte surface receptor involved in recognition and uptake of asialoglycoproteins. The role of CIG in hepatocyte attachment to collagen was investigated. Data are presented suggesting that this glycoprotein, which mediates the adhesion of fibroblasts to collagen, is not required for hepatocyte attachment to collagen.     

Increase of L-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh.

Hormonal regulation of L-serine dehydratase [L-serine hydro-lyase (deaminating), EC 4.2.1.13] was studied in primary cultures of adult-rat hepatocytes. The hepatocytes were isolated by collagenase perfusion and maintained in culture on collagen-gel/nylon-mesh substrata. L-Serine dehydratase activity was measured with [14C]threonine as substrate. The enzyme activity in hepatocytes of normal adult rats was low and declined rapidly in culture in L-15 medium containing 0.1 micro M-insulin and even more in the presence of glucose. L-Serine dehydratase activity in hepatocytes of rats with streptozotocin-induced diabetes was initially 20-fold higher than that of normal rats, but fell rapidly to a low value by 4 days in culture. Hormonal regulation of the enzyme activity was manifested by treatment of the cultured hepatocytes with insulin (0.1 micro M), glucagon (0.3 micro M), dexamethasone (10 micro M) and combinations of these hormones. Either glucagon or dexamethasone in the absence of insulin enhanced the activity of L-serine dehydratase, but failed to do so in the presence of insulin. Treatment with both hormones resulted in a 2-3-fold increase in enzyme activity in culture on days 3 and 4. Under conditions in which the enzyme activity was enhanced, glucose production by the cultured hepatocytes was concomitantly increased. Glucose production resulted in part from gluconeogenesis from pyruvate and not entirely from glycogenolysis. The gluconeogenic conditions of culture resulted in a decrease in cellular lipids in the cultured hepatocytes, as evidenced by ultrastructural studies.     

Primary cultures of rat hepatocytes in hollow fiber chambers

Summary Hepatocyte culture may represent an alternative to the use of animals to study drug detoxification by the liver. An ideal in vitro system should closely mimic the in vivo environment by providing continuous media perfusion and oxygenation, and should facilitate sampling of cells and culture media. To meet these criteria, a hollow fiber bioreactor seeded with isolated rat hepatocytes was developed and tested by measuring the formation of three products of the oxidative metabolism of diazepam and the glucuronidation of phenolsulfonphthalein (PSP). To compare the performance of conventional monolayer culture to that of the bioreactor system, diazepam metabolism was studied for 45 days in both systems. The oxygen dependency of diazepam metabolism was evaluated by perfusing the bioreactor in an oxygen-rich atmosphere (30%). Total diazepam metabolism was twofold higher in the O₂-rich perfused hollow fiber cultures than in the cultures perfused under normal conditions, reflecting an increase in temazepam and oxazepam production. Diazepam detoxification activity was significantly enhanced by oxygen (P≤0.001) over the life of the perfused cultures. PSP metabolism was similar in all three culture systems. By Day 10, diazepam metabolism in the oxygenated bioreactor system was 44% of the in vivo activity of rat hepatocytes. This activity dropped to 30% by Day 25 of culture. These results justify the use of perfused culture systems for in vitro detoxification studies as an alternative to animal use and emphasize the capacity of a culture device perfused under O₂-enriched conditions to maintain long-term P450 activity of rat hepatocytes.     

Phosphatidylinositol-dependent bond between alkaline phosphatase and collagen fibers in the periodontal ligament of rat molars

Alkaline phosphatase (ALP) is anchored to the outer leaflet of the lipid bilayer via phosphatidylinositol (PI) and ALP activity has been localized in the plasma membrane of numerous tissues. In the periodontal ligament ALP activity is found in the collagen fibers in addition to the plasma membrane of the osteoblasts and fibroblasts. In this study, we examined the distribution of ALP activity in the periodontal ligament of rat molars and also examined whether the bond between ALP and collagen fibers is dependent on PI by using phosphatidylinositol-specific phospholipase C (PI-PLC). ALP activity was distributed in the periodontal ligament. The activity mirrored the distribution of collagen fibers in the periodontal ligament. Cytochemical analysis also demonstrated that ALP activity was located not only in the plasma membrane of fibroblasts, but also in the collagen fiber bundles and fibrils in the periodontal ligament. After treatment with PI-PLC, the loss of ALP activity in the periodontal ligament was observed histochemically, and the loss of ALP activity in the fibroblasts as well as in the collagen fiber bundles and fibrils was observed cytochemically. These results strongly indicate that the bond between ALP and the collagen fibers is also dependent on PI.     

Regulation of collagen synthesis in fibroblasts within a three-dimensional collagen gel

Fibroblasts cultivated within a three-dimensional collagen gel display an elongated, spindle-like morphology, reduce their proliferation rate, contact the gel to a very dense tissue, and modify their metabolic activity as compared to monolayer cultures. Collagen synthesis measured as protein-bound hydroxyproline is reduced to 5% of the values found in monolayer culture. The reduction involving type I and type III collagen is due to decreased de novo synthesis and not to enhanced degradation. Dot blot hybridization, Northern blot analysis, and in situ hybridization using collagen I- and III-specific cDNA probes demonstrate that reduced biosynthesis rates are reflected by a marked reduction of pro alpha 1 (I), pro alpha 2 (I), and pro alpha 1 (III) collagen mRNA indicating pretranslational regulation. A similar reduction was observed for actin mRNA whereas levels of tubulin mRNA were similar for fibroblasts in monolayer culture or cultivated within the three-dimensional collagen gels. The data suggest a specific reprogramming of various cellular activities in response to contact with the reconstituted extracellular matrix.     

Contraction-relaxation of collagen fibers in LiBr-water-acetone solutions

Studies have been made of the contraction of collagen fibers in acetone due to the action of LiBr. No contraction occurs unless a small amount of water is added; on further increase of the water concentration, the fibers relax. The first contraction is irreversible. The effect of water is discussed in terms of hydrate formation.