1，2-二氯乙烷对大鼠肝细胞内游离钙离子浓度的影响

目的了解1,2-二氯乙烷染毒24h对大鼠肝细胞的损伤及其机理。方法运用显微荧光术测定了大鼠肝细胞内游离钙离子浓度,同时,测定了大鼠肝细胞培养上清液乳酸脱氢酶（LDH）活力作为大鼠肝细胞受损的指标。结果所有剂量组的1,2-二氯乙烷的大鼠肝细胞内游离钙离子浓度与对照组比较差异均无显著性（P〉0．05）;LDH活力仅在浓度为5mmol／L的1,2-二氯乙烷染毒组与对照组比较差异也无显著性（P〉0．05）;而1,2-二氯乙烷其他染毒组（即浓度为10mmol／L和20mmol／L）与对照组比较差异均有显著性（P〈0．01）。结论较高浓度（10mmol／L和20mmol／L）的1,2-二氯乙烷能损伤大鼠肝细胞,损伤的途径不是通过破坏肝细胞内钙稳态机制。     

Subcellular distribution and movement of 5''-nucleotidase in rat cells.

1. Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme, but over 75% inhibition of the cell-surface enzyme. 7. When surface-inhibited hepatocytes were washed and resuspended in buffer at 37 degrees C, 5'-nucleotidase was observed to redistribute from the intracellular pool to the cell surface.     

Cytotoxicity of colchicine derivatives in primary cultures of human hepatocytes

BACKGROUND: Colchicine has been used to treat gout for centuries. However, owing to its toxicity it displays a variety of side effects. The replacement of colchicine by less toxic but still active derivatives would solve this drawback. AIM: The aim of this study was to examine the cytotoxicity of 17 colchicine derivatives. METHODS: Primary cultures of human hepatocytes were the model of choice. Prior to testing, we measured the biochemical parameters of liver donors and the toxicological response of the hepatocytes cultures. For toxicity testing, cells were treated for 24 h with tested compounds in concentrations 1-100 microM. We monitored lactate dehydrogenase (LDH) leakage into the medium, mitochondrial activity (MTT test) and secretion of albumin. RESULTS: Our data show that LDH and MTT were less sensitive parameters compared to albumin secretion for monitoring the toxicity of colchicine derivatives. Compounds with lower antimitotic activity displayed lowered toxicity. CONCLUSION: Since human hepatocytes in culture are quiescent cells, they are not as susceptible to tropolone alkaloids as proliferating cells. Screening for colchicine derivatives with lowered cytotoxicity revealed that 10-O-demethylated compounds might be the substances of choice.     

Toxic Effects of Tacrine on Primary Hepatocytes and Liver Epithelial Cells in Culture

Administration of tacrine (THA) for the treatment of Alzheimer's disease results in a reversible hepatotoxicity in 30–50% of patients, as indicated by an increase in transaminase levels. However, the intracellular mechanisms underlying such a toxicity have not yet been elucidated. In this study, we performed short-term and long-term in vitro treatments on primary human and rat hepatocyte cultures as well as on nonparenchymal rat liver epithelial cells (RLEC), known as CYP1A-deficient cells. Cell ultrastructure was analyzed under different conditions and the release of lactate dehydrogenase (LDH) was used to evaluate cytotoxicity. The effects of THA on protein synthesis, intermediary metabolism and reduced glutathione (GSH) level were also determined in rat hepatocytes. THA induced dose-dependent toxic effects in liver parenchymal and nonparenchymal cells, with human hepatocytes being less sensitive. This toxicity appeared to be unrelated to metabolism of THA since similar effects were observed in rat hepatocytes and RLEC, in which THA metabolism was found negligible. Ribosome aggregation appeared only at high concentrations (>1 mmol/L) and was not specific to hepatocytes. Therefore, the THA-induced decrease in protein synthesis observed at lower concentrations was likely not related to this alteration. ATP and glycogen levels as well as GSH content were reduced upon THA. However, while glycogen level decreased at THA doses similar to those inducing an increase in LDH release, the fall in ATP and GSH contents occurred at higher doses. Thus, glycogen level in hepatocytes appeared to be a more sensitive indicator of THA toxicity than were ATP and GSH levels. We also found that protein synthesis started to decrease at THA doses that were still ineffective on LDH release. This might suggest that the decrease in synthesis of one or several proteins upon THA treatment represents the early signal leading cells to death.     

Intracellular lactate dehydrogenase concentration as an index of cytotoxicity in rat hepatocyte primary culture

In searching for a reliable index for cytotoxicity testing in rat hepatocyte primary culture, lactate dehydrogenase (LDH) concentrations in lysates of attached hepatocytes and LDH released into the culture medium were compared under conditions of exposure to various dosages of sodium chloride, sodium salicylate, R-warfarin, acetaminophen, phenylbutazone, and furosemide (frusemide). The amount of intracellular LDH was assessed by inducing the cells to release the enzyme with 0.1% Tritron X-100. The induced LDH leakage was completed in 1 hr and the LDH activity was stable in storage at 10° for 2 weeks. We found that intracellular LDH is a direct indicator of the number of viable hepatocytes in contrast to the LDH released, because released LDH does not account for the significant number of cells detached from monolayer but which are not leaky, during the 6-hr test period. Based on IC₅₀ values (50% inhibitory concentration), the relative cytotoxicities are R-warfarin > phenylbutazone > furosemide > acetaminophen > sodium salicylate > sodium chloride.Abbreviations DMSO dimethyl sulfoxide - HPC hepatocyte primary culture - IC₅₀ 50% inhibitory concentration - LDH lactate dehydrogenase     

The effect of EGF and the comitogen, norepinephrine, on the proliferative responses of fresh and cryopreserved rat and mouse hepatocytes

The effect of cryopreservation on the proliferative response of fresh and cryopreserved (CP) rat and mouse hepatocytes was studied. Of the parameters measured, incorporation of 3H-thymidine and bromodeoxyuridine (BdrU) incorporation were the most sensitive and LDH content was the least sensitive. The optimal seeding density for epidermal growth factor (EGF)-stimulated proliferative response in fresh rat and mouse hepatocytes was 1.8 x 10(4) cells/cm2 and 2.1 x 10(4) cells/cm2, respectively. 3H-thymidine incorporation by fresh rat and mouse hepatocytes was maximal in cultures treated with 10 and 5 ng/ml EGF, respectively. The cell attachment of fresh rat hepatocytes after 48 h was higher (68%) than CP (42%), therefore, the CP hepatocyte seeding density was increased to 7.1 x 10(4) cells/cm2 so that the cell number after 48 h was the same as fresh hepatocytes. Using the adjusted seeding density, the 3H-thymidine and BdrU incorporation into fresh and CP rat hepatocytes was equivalent. The attachment efficiencies of fresh and CP mouse hepatocytes were the same, therefore, no adjustment was needed. The proliferative response (3H-thymidine incorporation and DNA content) to EGF was the same in fresh and CP mouse hepatocytes. The comitogen, norepinephrine (NE), increased the proliferative response to EGF to the same extent in both fresh and CP rat hepatocytes. In summary, cryopreserved rat and mouse hepatocytes retain their ability to proliferate in culture. Adjustment and monitoring of the seeding density is of high importance, especially with rat hepatocytes, which lose some attachment capacity after cryopreservation. The secondary mitogenic effect of NE is also retained by cryopreserved rat hepatocytes, suggesting that these cells retain alpha1-receptor function.     

The in vitro biological activity of Lepidium meyenii extracts

The biological activity of methanolic and aqueous extracts from dehydrated hypocotyls of Lepidium meyenii (Brassicaceae, vernacular name “maca”), was studied on rat hepatocytes and human breast cancer MCF-7 cells. The extracts did not exhibit cytotoxicity in hepatocyte primary cultures up to 10 mg/ml as measured by the MTT viability test, and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) leakage. Moreover, after 72 h, extracts inhibited LDH and AST leakage from the hepatocytes. When hepatocytes were intoxicated by t-butyl hydroperoxide, neither extract prevented oxidative damage. Both extracts showed weak antioxidant activity in the DPPH radical scavenging test with IC₅₀ values of 3.46 ± 0.16 and 0.71 ± 0.10 mg/ml, for aqueous and methanolic extracts, respectively. Thus, the observed effect on spontaneous enzyme leakage is probably mediated through mechanisms other than antioxidant activity. Both methanolic and aqueous extracts have shown estrogenic activity comparable with that of silymarin in MCF-7 cell line. Maca estrogenicity was exhibited in the range from 100 to 200 μg of extract per ml. The findings in the present study show that maca does not display in vitro hepatotoxicity. In contrast, a slight cytoprotective effect, probably not mediated by antioxidant capacity, was noted. Maca extracts exhibited estrogenic activity comparably to the effect of silymarin in MCF-7 cells.     

Radioimmunoassay of metallothionein in rabbit,rat, mouse,Chinese hamster,and human cells

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.     

The isolation of endosome-derived vesicles from rat hepatocytes.

Intracellular 5'-nucleotidase involved in membrane circulation in rat hepatocytes is latent, and is protected from inhibition when whole cells are incubated with inhibiting antiserum at 2 degrees C [Stanley, Edwards & Luzio (1980) Biochem. J. 186, 59-69]. These two criteria were used to identify intracellular membrane vesicles containing 5'-nucleotidase on Ficoll density gradients. A sharply defined turbid band containing intracellular 5'-nucleotidase isolated on density gradients was further fractionated by immunoadsorption of plasma-membrane fragments derived from the cell surface of surface-inhibited cells on to an anti-(immunoglobulin G) immunoadsorbent. The resulting non-adsorbed membrane fraction consisted of vesicles of uniform size (approx. 65 nm diam.), but was not identifiable as any known organelle. This fraction could account for approx. 5% of the total cell 5'-nucleotidase activity, and the enzyme activity measured was 55% latent. The fraction had a restricted polypeptide composition but similar phospholipid composition compared with plasma membrane. We suggest that the vesicles observed in this fraction were derived from the endocytic pathway.     

Use of electroporation to introduce biologically active foreign genes into primary rat hepatocytes.

A method is described for introducing and expressing cloned genes in isolated hepatocytes. Primary rat hepatocytes isolated by collagenase perfusion were transfected in suspension with plasmid pSV2CAT by electroporation. Forty-eight hours later, soluble extracts from transfected hepatocytes showed chloramphenicol acetyltransferase activity comparable to that obtained in rat hepatoma cell line H4AzC2 by calcium phosphate or DEAE-dextran transfection. The latter two methods could not be used successfully for primary hepatocytes because of cytotoxicity of these reagents. This indicates that electroporation is a useful method to obtain transient expression of foreign genes in primary epithelial cells, such as rat hepatocytes, which are difficult to maintain in cell culture.     

Uracil-DNA glycosidase studied with an immune serum

Immune antiserum to uracil-DNA glycosylase was obtained by immunizing rabbits with an enzyme isolated from the rat liver. Antiserum was found to suppress the activity of uracil-DNA glycosylase not only in the extracts of rat liver, but also in the extracts of brain, cardiac muscle, kidney, spleen, thymus of rats, and in those of human placenta too. This enables us to make a conclusion about the similarity in antigenic properties of the enzyme in cells of various types of differentiation. Indirect immunofluorescent test shows a slight staining of the periphery of the nucleus in normal liver hepatocytes and the intensive staining of the inner part of the nucleus in hepatocytes of regenerating liver. Therefore it is concluded that the enzymatic activity increases as cells proliferate. This may be the result of the appearance of uracil in DNA during replication.     

Long-term culture of adult rat hepatocyte spheroids

Hepatocytes from adult rats were cultured on poly-HEMA-coated surface to form spheroids in hormonally defined media as previously shown with newborn rat hepatocytes. Spheroidal aggregates of adult rat hepatocytes were morphologically similar to those of newborn rat hepatocytes and could also form a monolayer of uniform liver parenchyma-like cells when transferred on collagen-coated surfaces even after 2 months of culture. Under these culture conditions, albumin and transferrin secreted in vitro by adult rat hepatocyte spheroids were detectable by immunoprecipitation method at least until 2 months of culture. The production of proteins by hepatocyte spheroids could be regulated in vitro by IL-6: the secretion of alpha 2-macroglobulin was increased and the secretion of albumin was decreased in the presence of this cytokine. In addition, cytochrome P450 IA1 was strongly induced by methylcholanthrene in adult rat hepatocyte spheroids, and the induction remained relatively constant up to 22 days of culture. These cells were also able to metabolize lidocaine to monoethylglycinexylidine when measured up to 14 days of culture, showing the presence of a relatively high level of P450 IIIA2. The UDP-glucuronyltransferase activity, specific for bilirubin conjugation, decreased to 18% of the initial value after 2 weeks of culture. This work showed that adult rat hepatocytes in long-term spheroid culture kept differentiated functions, providing a new model for the in vitro study of hepatocyte functions and complementing that of newborn rat hepatocytes using the same system.     

A simplified model of hypoxic injury in primary cultured rat hepatocytes

Summary The Anaeropack system for cell culture, which was originally designed for the growth of anaerobic bacteria, was used to produce a hypoxic atmosphere for cultured hepatocytes. We measured changes in the oxygen and carbon dioxide concentrations and the atmospheric temperature in an airtight jar. We also measured changes in the pH of the medium during hypoxia to assess the accuracy of this system. Moreover, we used three durations (2, 3, and 4 h) of hypoxia and 8 h of reoxygenation in cultured rat hepatocytes, and then measured the lactate dehydrogenase (LDH), ketone body concentration (acetoacetate + β-hydroxybutyrate), and the ketone body ratio (KBR: acetoacetate/β-hydroxybutyrate) in the medium in order to assess the suitability of this system as a model for reperfusion following liver ischemia. The oxygen concentration dropped to 1% or less within 1 h. The concentration of carbon dioxide rose to about 5% at 30 min after the induction of the hypoxic conditions, and was maintained at this level for 5 h. No effect of the reaction heat produced by the oxygen absorbent in the jar was recognized. The extent of cell injury produced by changing the hypoxic parameters was satisfactorily reflected by the KBR, the ketone body concentration, and the LDH activity released into the medium. Because this model can duplicate the conditions of the hepatocytes during revascularization following ischemic liver, and the Anaeropack system for cell culture is easy to manipulate, it seems suitable for the experimental study of hypoxic injury and revascularization in vitro.     

Comparative subcellular distribution of benzaldehyde and acetaldehyde dehydrogenase activities in two hepatoma cell lines and in normal hepatocytes.

The NAD- and NADP-dependent aldehyde dehydrogenase (ALDH) activities were evaluated in two rat hepatoma cell lines, namely the well-differentiated MH1C1 line and the less differentiated HTC line. Each activity was determined in parallel in isolated rat hepatocytes, for comparison. The aliphatic aldehyde acetaldehyde (ACA) and the aromatic aldehyde benzaldehyde (BA) were used as substrates. With the first substrate the ALDH activities found in the crude cytoplasmic extracts were lower in hepatoma cells than in normal hepatocytes, especially when measured with NADP as coenzyme (ACA/NADP). Otherwise, with benzaldehyde as substrate the NAD-dependent enzyme activity (BA/NAD) was increased about 9-fold in HTC cells over hepatocytes and decreased in MH1C1 cells, while the NADP-dependent (BA/NADP) activity was increased 38- and 2.5-fold in HTC and MH1C1 cell lines, respectively. Studies on the subcellular distribution of these enzyme activities showed that the activity measured with acetaldehyde and NAD (ACA/NAD) was almost equally distributed between the cytosol and the subcellular particles in the three cell populations, but the ACA/NADP activity was shifted towards the cytosolic compartment in hepatomas, especially in HTC cells. The BA/NAD and BA/NADP ALDH activities found in the organelles of hepatoma cells were markedly reduced in comparison with hepatocytes, in favour of the cytosol. The most striking difference between the normal and the transformed cells was the 94-fold increase over hepatocytes of the BA/NADP activity, found in the cytosolic fractions of HTC cells. MH1C1 cells showed a less pronounced (7.5-fold) enhancement of this tumour-associated specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenylate cyclase and phosphodiesterase activities in rat hepatocytes cultured in the presence and absence of dexamethasone

The effects of glucagon and dexamethasone on the activities of the enzymes involved in cyclic adenosine 3':5'-monophosphate (cyclic AMP) metabolism in primary monolayer cell cultures of adult rat hepatocytes were examined. Short-term experiments indicated that the magnitude of the cultured cells' response to glucagon, as measured by production of cyclic AMP, was essentially the same as that for freshly isolated hepatocytes. However, the time course of this response was markedly different. Although the activity of adenylate cyclase is maintained throughout the culture period at a level similar to that of the freshly isolated hepatocytes, the activity of both low and high Km forms of phosphodiesterase decreases rapidly with length of time in vitro. This is reflected by an increase in cyclic AMP produced in response to glucagon and theophylline by cells of different ages. Dexamethasone caused an increased loss of phosphodiesterase activity, as well as increased cyclic AMP accumulation in the presence or absence of theophylline. Various agents failed to restore the lost phosphodiesterase activity. These results may indicate that phosphodiesterase activity is more sensitive to the inevitable inadequacies of the in vitro environment of cultured hepatocytes than adenylate cyclase. It was also found that a modification of the method of Seglen (1) for the preparation of isolated hepatocytes yielded cells that had less phosphodiesterase activity than those prepared by the method of Berry and Friend (2).     

Hormonal effects on the biosynthesis of lactate dehydrogenase in rat hepatocytes.

Rat hepatocytes have been studied in suspension culture for 10-h periods. Levels of extractable lactate dehydrogenase (LDH) have been measured in these hepatocytes at hourly intervals in order to note the balance between biosynthesis and degradation of this enzyme. Newly synthesized LDH has been measured by following the rate of incorporation of [3H]leucine into radiochemically pure LDH of high specific catalytic activity as isolated by a rapid affinity chromatographic procedure. The effects of the addition of physiological concentrations of the following hormones at the beginning of 10-h culture periods immediately following preparation of the hepatocytes by the collagen perfusion procedure have been recorded. The hormones triiodothyronine (T3), insulin, glucagon, and dexamethasone have been added singly or in combination. The culture medium has supplied variable amounts of these hormones in the 10% of fetal calf (or other) serum added, and the hepatocytes themselves have provided intracellular amounts of hormones. In addition to the added hormones, N6,O2'-dibutyryl cyclic AMP (Bt2cAMP) has also been studied. Control suspensions of hepatocytes show reproducible initial levels of extractable LDH which are maintained or slightly increased during 10 h. Such control systems also incorporate [3H]leucine into total protein and into highly purified LDH at reproducible rates during 10 h of incubation. The effects of added hormones on LDH lavels are as follows: (a) T3 causes about a 2-fold increase in LDH at 7 to 8 h in hepatocytes from young adult animals, an effect which is lowered in either younger or older animals or in thyroidectomized animals. (b) Insulin leads to a similar increase in LDH at 5 to 6 h and a falling off at 8 to 10 h. (c) Glucagon also causes an approximate doubling of the amount of extractable LDH during a 10-doubling of the amount of extractable LDH during a 10-h period. (d) Dexamethasone does not produce an increase. (e) Bt2-cAMP produces an effect indistinguishable from that of glucagon. Paired combinations of these hormones fail to produce an additive response in any case. The combinations of T3 plus dexamethaseon and insulin plus dexamethasone lead to significant reductions in levels of extractable LDH when compared to the single hormone effects cited above. With respect to rates of synthesis of total protein as measured by [3H]leucine incorporation, only glucagon, glucagon plus Bt2-cAMP, glucagon plus insulin, T3 plus Bt2cAMP, and T3 plus insulin produce significant increases during a 10-h period. However, when [3H]leucine incorporation into highly purified LDH is measured as an index of LDH biosynthesis, T3, insulin, and glucagon consistently increase the biosynthetic rates during a 10-h period. Bt2cAMP produces a smaller increase. Dexamethasone fails to produce any significant change when compared to controls. Paired combinations of hormones again do not produce any additive effect on LDH biosynthesis when the hormone producing the higher level is taken as the reference...     

Intermittent Anoxia Reduces Oxygen Free Radicals Formation During Reoxygenation in Rat Hepatocytes

The sensitivity of liver cells to anoxia is a major problem afflicting liver preservation and transplantation. Intermittent ischemia has been proposed to reduce reperfusion injury. The aim of the study was to assess oxygen free radical formation and cell injury during continuous or intermittent anoxia/reoxygenation in rat hepatocytes. Anion superoxide was measured by lucigenin-enhanced chemiluminescence and cell damage by LDH release and trypan blue uptake. During anoxia, superoxide generation dropped to background level in both groups; trypan blue uptake and LDH release, which increased progressively, were significantly greater in hepatocytes exposed to continuous compared to intermittent anoxia. During reoxygenation, a massive generation of superoxide anion formation, followed by a sharp increase in LDH release, was observed in both groups. However, both oxyradical generation and cell injury were significantly greater in cells exposed to continuous compared to intermittent anoxia. The data, showing that intermittent oxygen deprivation reduce liver cell injury and oxygen free radical formation determined by anoxia/reoxygenation, suggest a novel possible approach to the reduction of reperfusion injury.     

The effect of pH on the viability of hypothermically stored rat hepatocytes

Hepatocytes isolated from the rat liver were stored for up to 72 hr at 4 degrees C in a tissue culture medium (Liebovitz-15) at different pH values to determine how pH affects hepatocyte viability. This is a model to simulate cold storage of livers for transplantation and determine the optimal pH for maintenance of liver cell function. The cells were stored in the absence of oxygen. At the end of cold storage the percentage of the total cellular LDH released into the extracellular medium was used as a measure of hepatocyte viability. Also, lactate dehydrogenase (LDH) release was determined in hepatocytes incubated at normothermia (37 degrees C) for 90 min following 72 hr of cold storage. The results demonstrate that hepatocytes tolerate a wide range of pH values in the storage medium and that only about 10% of the total LDH was released from hepatocytes stored up to 72 hr at pH's from 5.0 to 8.0. Normothermic incubation, however, demonstrated that the pH of the storage medium affected viability. After 48 hr of storage only hepatocytes stored at pH values from 7.0 to 8.0 remained viable (LDH release similar to that of freshly incubated hepatocytes = 28 +/- 7.2%). After 72 hr of storage and 90 min of normothermic incubation, hepatocytes incubated at all pH values studied were nonviable (greater than 60% release of LDH). These results suggest that the optimal pH for storage of hepatocytes at 4 degrees C is near neutrality (7.0 to 7.4).     

Immunocytochemical localization of alpha-fetoprotein in the developing and carbon tetrachloride-treated rat liver.

The immunocytochemical localization of alpha-fetoprotein (AFP)-producing cells was observed in pre- and postnatal and carbon tetrachloride (CCl4)-treated rat livers in comparison with that of albumin (ALB)-producing cells. According to immunoblotting data, considerable numbers of AFP-positive hepatocytes were observed in the differentiating liver between prenatal day 19 and postnatal day 0 (6 h after birth). Analyses by serial section profiles of these cells revealed that certain AFP-positive hepatocytes are also stained with ALB antiserum. Immunoelectron microscopy of the AFP-producing cells revealed that immunoreactive gold particles are preferentially localized in rough endoplasmic cisternae, Golgi apparatus and Golgi-derived vesicles near the cell surface. In addition, the release of the content of the Golgi-derived vesicles into the differentiating bile canaliculi as well as into the space of Disse by exocytosis is apparent. In CCl4-treated rat liver, immunoreactions to AFP are localized exclusively in newly formed hepatocytes of the regenerative tissue. These AFP-positive cells have not established the hepatic cell cords, and the adjacent ones are conjugated to each other mainly by simple attachment devices as in the case of those in pre- and postnatal rat liver.     

Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes—Freshly isolated cells,cell suspensions,and primary monolayer cultures

Summary Leakage of lactate dehydrogenase and staining by the vital dye trypan blue were investigated in adult rat hepatocytes at the time of isolation, in suspensions up to 3 h and in primary monolayer cultures up to 3 d. These two parameters of plasma membrane integrity were found to correlate closely in hepatocyte suspensions, but to a lesser degree in monolayer cultures. Functional activity was demonstrated in culture by glucose consumption and lactic acid production. There was a balance of total lactate dehydrogenase (LDH) activity over time for both hepatocyte suspensions and cultures. Loss of LDH activity in the cell fraction was accompanied by a corresponding increase in enzyme activity in the media fraction. Lactate dehydrogenase activity per dye-excluding hepatocyte was calculated to be 9.2±1.5×10⁻⁶ IU assayed at 37°C for 25 preparations of isolated hepatocytes. The results suggest that leakage of cytoplasmic enzyme and vital dye staining are of comparable sensitivity in evaluating hepatocyte preparations. Measurement of LDH leakage offers a less subjective alternative to cell counting procedures and is applicable to both attached and suspended cells. This study was supported in part by Grants HL-11945-11 and 1-RO1-AM 26520-01A1 from the National Institutes of Health, Bethesda, MD.