Cytotoxicity of bile in human Hep G2 cells and in primary cultures of rat hepatocytes

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.     

Sulfotransferase gene expression in primary cultures of rat hepatocytes

Hepatocyte cultures have been used in pharmacotoxicological studies, and sulfotransferases (ST) are important drug-metabolizing enzymes in liver. The expression of sulfotransferases in hepatocyte cultures has not been examined systematically. In the present study, the mRNA levels of different sulfotransferases in male and female rat hepatocytes were examined by northern-blot analyses. Various culture conditions such as different matrices (collagen, matrigel, collagen sandwich, or co-culture with epithelial cells), medium (Way-mouth's MB 752/1 and Modified Chee's Medium) and glucocorticoid supplementation (dexamethasone, 0.1 microM) were compared. Phenol ST (ST1A1) mRNA levels decreased to about 50% of initial mRNA levels within 10 hr of culture. At 96 hr, ST1A1 mRNA levels were approximately 20% of initial values when cultured on collagen, matrigel or co-culture. The two media did not differ in ability to maintain ST1A1 mRNA levels in the absence of dexamethasone (DEX); however, DEX addition to either medium resulted in ST1A1 mRNA levels greater than 100% of the initial mRNA levels at 96 hr, with the greatest increase observed using the matrigel substratum and Chee's medium. In the absence of DEX, the mRNA levels of N-hydroxy-2-acetylaminoflurene sulfortransferase (ST1C1), estrogen sulfotransferase (ST1E2) and hydroxysteroid sulfotransferase (ST-20/21, ST-40/41, ST-60) fell to approximately 20% of their initial levels within 24 hr, and to less than 5% at 96 hr. The loss of expression of these sulfotransferases was observed with all culture conditions. Addition of DEX to the media resulted in ST-40/41 and ST-60 mRNA expression at 20 and 35% of their initial values, respectively, in cultures maintained on matrigel and Chee's medium at 96 hr. These data suggest that sulfotransferases lose their constitutive expression in hepatocyte culture, but retain their inducibility.     

Interactions between metallothionein inducers in rat liver and primary cultures of rat hepatocytes

The interaction of Zn, stress and endotoxin on liver metallothionein (MT) regulation has been studied in the rat. Zn, stress and endotoxin increased liver MT levels significantly, by 12-, 5- and 8-fold, respectively. The previous administration of Zn to stress or endotoxin treatments increased MT levels by 35- and 42-fold, respectively, indicating a synergistic effect in both cases. In contrast, when liver MT was preinduced by stress, MT levels were further increased by endotoxin only in an additive manner. In another experiment where liver MT induction by stress was studied in control rats and in rats with preinduced MT by Zn, endotoxin or stress, it was found that Zn pretreated animals had higher MT-I mRNA levels than endotoxin- or stress-pretreated ones. No synergisms between dexamethasone, Zn, TNF and IFN were observed in primary culture of hepatocytes. These results suggest that the observed synergisms between Zn and other MT inducers in vivo in the liver is a consequence of increased Zn levels in the body and mobilization capacity, with concomitant MT synthesis.     

Osmotic regulation of the heat shock response in primary rat hepatocytes

The crystal structure of recombinant glycosylasparaginase from Flavobacterium meningosepticum has been determined at 2.32 angstroms resolution. This enzyme is a glycoamidase that cleaves the link between the asparagine and the N-acetylglucosamine of N-linked oligosaccharides and plays a major role in the degradation of glycoproteins. The three-dimensional structure of the bacterial enzyme is very similar to that of the human enzyme, although it lacks the four disulfide bridges found in the human enzyme. The main difference is the absence of a small random coil domain at the end of the alpha-chain that forms part of the substrate binding cleft and that has a role in the stabilization of the tetramer of the human enzyme. The bacterial glycosylasparaginase is observed as an (alphabeta)2-tetramer in the crystal, despite being a dimer in solution. The study of the structure of the bacterial enzyme allows further evaluation of the effects of disease-causing mutations in the human enzyme and confirms the suitability of the bacterial enzyme as a model for functional analysis.     

Ribavirin inhibits protein synthesis and cell proliferation induced by mitogenic factors in primary human and rat hepatocytes

Ribavirin, a guanosine analog, used in combination with interferon alpha (IFN-alpha) in the treatment of chronic hepatitis induced by hepatitis C virus (HCV) infection, has been shown to improve liver histology and to decrease transaminases even when administered alone. We analyzed the direct effects of ribavirin on the liver by using primary cultures of human and rat hepatocytes. Between 10 to 60 micromol/L, ribavirin was found to inhibit both the synthesis and secretion of whole proteins in a time- and dose-dependent fashion. Such an effect was confirmed by the measurement of albumin and haptoglobin secretion rates. [3H]-Thymidine incorporation was suppressed both in hepatocyte growth factor-stimulated human hepatocytes and in epidermal growth factor (EGF)-stimulated rat hepatocytes in the presence of ribavirin. The inhibitory effect on DNA synthesis was associated with a delayed progression to S phase of the cell cycle, as determined by flow cytometry and detection of cyclin A and cdc2 which are two proteins expressed during the S phase. The inhibition of DNA synthesis, caused by 50 micromol/L ribavirin, was completely restored by the addition of 80 micromol/L guanosine. These observations demonstrate that ribavirin at concentrations close to those found in plasma of treated patients can directly affect hepatic functions in vitro. Its effects could, however, be reduced in vivo by guanosine salvage supply.     

Regulation of rat hepatic 3 alpha-hydroxysteroid dehydrogenase in vivo and in primary cultures of rat hepatocytes

Lamoids in North America harbor a wide variety of parasites. Treatment and control methods based on previous experience with parasites of cattle and sheep have been successful, but problems do exist. First, the pharmacokinetics for most anthelmintics have not been evaluated in llamas. Second, even though llamas, sheep, and cattle share many parasites, the two most common nematodes found in llamas (C. mentulatus and T. tenuis) are not part of the parasitic fauna of livestock. This presents difficulties in basing treatment and control methods on those recommended for cattle and sheep. Variability in host response to the same parasite also hinders the use of cattle and sheep as models for the llama. This is best demonstrated by F. magna and F. hepatica; the reaction induced by the first more closely resembles those seen in cattle than sheep, but the reaction induced by the second more closely resembles those seen in sheep than cattle. Finally, parasites known to be pathogenic in livestock (e.g., N. battus) have unknown effects in llamas. These examples illustrate that we must use caution when extrapolating existing knowledge regarding the parasites of sheep and cattle to llamas. Further research on the epidemiology of parasites peculiar to the llama is needed to enhance control efforts. Improved methods of diagnosis and treatment of parasites also are areas in which further efforts are needed.     

Effects of curcumin on P-glycoprotein in primary cultures of rat hepatocytes

Curcumin is a natural phenolic compound found in the rhizomes of Curcuma longa and endowed with beneficial biological activities including antioxidant, anticarcinogenic and hepatoprotective effects. In this study curcumin was tested for its potential ability to interact in vitro with hepatic P-glycoprotein (Pgp), in a model system represented by primary cultures of rat hepatocytes, in which spontaneous overexpression of multidrug resistance (mdr) genes occurs. In both freshly-plated hepatocytes, containing low levels of Pgp, and 72 hour-cultured hepatocytes, containing high levels of Pgp, the Rhodamine-123 (R-123) efflux, which represents a specific functional test for Pgp-mediated transport, was inhibited by curcumin in a dose-dependent manner. Western blot analysis showed that 25microM curcumin, when included in the culture medium throughout the experimental observation (72 hours), was able to significantly lower the increase of mAb C219-immunoreactive protein spontaneously occurring in the cells during culture. Curcumin, at doses ranging from 50 to 150microM was cytotoxic for freshly-plated hepatocytes, as shown by the strong decrease in the cell ability to exclude trypan blue 24 hours later, but it was significantly less cytotoxic when added to 24 or 48 hour-cultured cells. The resistance to curcumin, progressively acquired by cells during culture, was significantly reduced by high concentrations of dexamethasone (DEX) or dimethyl-sulfoxide (DMSO), culture conditions known to inhibit the spontaneous overexpression of Pgp. In addition, in a concentration-dependent manner, verapamil reverted curcumin resistance in Pgp overexpressing hepatocytes. In photoaffinity labeling studies, curcumin competed with azidopine for binding to Pgp, suggesting a direct interaction with glycoprotein. These results suggest that curcumin is able to modulate in vitro both expression and function of hepatic Pgp and support the hypothesis that curcumin, a chemopreventive phytochemical, could reveal itself also as a compound endowed with chemosensitizing properties on mdr phenotype.     

Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity. The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes. By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [3H]thymidine injection it was found that the label moved largely into the tetraploid compartment. Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation. The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.     

Oxidative damage and fumonisin B1-induced toxicity in primary rat hepatocytes and rat liver in vivo

The particle size distribution and the metal speciation of the heavy metals were investigated on dredged sediment and on the fractions obtained by mechanical agitated (Denver) flotation. The transition metal ions (cadmium, copper, lead and zinc) were flotated specifically independent of the particle size. Particle size analysis, EDTA extraction and sequential extracts indicated that during flotation a redistribution of metals occurred due to the oxidation of metal sulphides. This oxidation process was more pronounced when the flotation was performed at higher pH values and resulted in a decrease in flotation specificity.     

Enhanced proliferation of fetal rat hepatocytes in primary culture induced by ritodrine

OBJECTIVE: Although ritodrine crosses the placenta, its direct effect on fetal cell proliferation has not been reported. We hypothesized that beta 2-adrenergic receptor stimulation could promote fetal liver growth. STUDY DESIGN: Ritodrine was added to serum- and hormone-free primary cultures of fetal, neonatal, or adult rat hepatocytes. We measured both tritiated thymidine incorporation into deoxyribonucleic acid and nucleus number. The effect of ritodrine on cell cycle was also analyzed with flow cytometry. RESULTS: Ritodrine enhanced the proliferation of fetal rat hepatocytes. Ritodrine remarkably stimulated deoxyribonucleic acid synthesis of fetal and neonatal but not adult hepatocytes. The effect was dose dependent and was antagonized by propranolol. Analysis of the nuclear deoxyribonucleic acid content derived from flow cytometry revealed that cells stimulated by ritodrine entered S phase. CONCLUSION: These results indicate that ritodrine may promote the proliferation of fetal hepatocytes through the stimulation of beta 2-adrenergic receptors, followed by induction of deoxyribonucleic acid synthesis.     

Hormonal regulation of aldolase B gene expression in rat primary cultured hepatocytes

A high performance liquid chromatography method for the determination of N3-methyl-5'-deoxy-5-fluorouridine, a possible metabolic product of the anticancer pro-drug 5'-deoxy-5-fluorouridine, in human serum and urine is described. Sample treatment involved addition of internal standard (5-bromouracil) and protein precipitation with ammonium sulphate (serum samples) followed by liquid-liquid extraction with ethyl acetate-isopropanol (90:10, v/v). The average recovery at 0.5 mg ml-1 level was (80 +/- 4%). A linear response extending over two decades of concentration was observed. Detection limits of 50 and 100 ng ml-1 were obtained in serum and urine, respectively.     

Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells

Symptomatic and asymptomatic astrovirus infection was prospectively determined in a 3-year birth cohort of Mayan infants. Stool samples from 271 infants and 268 older siblings were tested for astrovirus, adenovirus 40/41, rotavirus and Salmonella, Shigella and Campylobacter species. Concurrent diarrhea, vomiting, fever, or anorexia were noted. Astrovirus was detected in 164 infants (61%) and 20 siblings (7%). Rotavirus (4%) and adenovirus 40/41 (13%) were isolated less frequently. Of all diarrheal episodes reported at a visit, 26% (78/305) were associated with astrovirus; 17% (78/452) of astrovirus infections were associated with diarrhea and 9% with other symptoms. Only diarrhea was associated with astrovirus infection (odds ratio, 1.4; 95% confidence interval [CI], 1.07-1.92; P = .01). Of infants with astrovirus, 70% shed at multiple visits over a period of 2-17 weeks (median, 5). The point prevalence of astrovirus infection was significantly higher among infants than siblings (relative risk, 6.18; 95% CI, 3.93-9.72; P < .0001, chi2). Astrovirus was identified throughout the year, peaked in March and May, and decreased in September. In this population, astrovirus was the most common enteric pathogen isolated; symptomatic infection was prevalent among infants.     

Occurrence and possible consequences of multipolar mitoses in primary cultures of adult rat hepatocytes

Proliferating primary cultures of adult rat hepatocytes are characterized by the occurrence of multipolar mitoses, and chromosome loss resulting in the formation of micronuclei at telophase. The percentage of multipolar mitotic figures was determined to be 12.76 +/- 7.9%, 80% of which were tripolar. Multipolar mitotic stages showed a high incidence of chromosome loss, increasing from meta- (61.7 +/- 16.6%) to telophase (72.1 +/- 19.3%). Regular bipolar mitotic figures on the other hand also showed chromosome loss, however, to a lesser degree and decreasing from meta- (49.5 +/- 10.4%) to telophase (34.9 +/- 7.9%). The incidence of chromosome loss even in regular mitotic figures is very high compared to other cells and appears to depend on another special feature of hepatocytes: they remain flat and well attached during mitosis, so that shearing forces could be responsible for the separation of chromosomes from the mitotic spindle. Additionally this morphology creates a situation allowing for a maximal interaction of mitotic spindles of binucleated cells, leading to the high rate of multipolar mitoses observed. Both multipolar mitoses and chromosome loss could also explain the consecutive detachment of hepatocytes reported for proliferating primary cultures, since the aneuploid daughter cells generated can be expected to be non-viable in most cases and eventually detach.     

Comparison of tacrine-induced cytotoxicity in primary cultures of rat, mouse, monkey, dog, rabbit, and human hepatocytes

Cisplatin (DDP) is currently one of the most effective drugs for the treatment of cancer. It causes primarily intrastrand DNA-DNA cross-links, and is highly mutagenic and carcinogenic in both in vitro and in vivo experimental models. There is, however, considerable variability between the response seen in different cellular systems, probably at least partly because of the different cellular DNA repair capacities. A number of analogues of cisplatin have been developed and one of these, carboplatin (CDDCA), is also in widespread clinical use. Although it is somewhat less toxic, there is no evidence that its mode of action differs from that of cisplatin. A limited amount of mutagenicity data suggests that it has similar mutagenic and carcinogenic consequences as the parent drug. Many further analogues of cisplatin are now in clinical trials, and some of these appear to have different DNA repair responses (and therefore possibly the development of clinical resistance). Although some (e.g., iproplatin and spiroplatin) are less mutagenic than either cisplatin or carboplatin, these appear to be the ones least likely to achieve wide use. There are insufficient data on several of the most promising clinical analogues (e.g., DWA2114R and ACDDP) to judge their relative mutagenic and carcinogenic potential. Detailed studies on the DNA repair and mutagenicity characteristics of these compounds will not only provide clinically relevant data, but may also aid in the selection of further useful antitumour agents in this series.     

Metabolic transformations of leukotriene B4 in primary cultures of human hepatocytes

Analytic expressions for plasma total titratable base, base excess (DeltaCB), strong-ion difference, change in strong-ion difference (DeltaSID), change in Van Slyke standard bicarbonate (DeltaVSSB), anion gap, and change in anion gap are derived as a function of pH, total buffer ion concentration, and conditional molar equilibrium constants. The behavior of these various parameters under respiratory and metabolic acid-base disturbances for constant and variable buffer ion concentrations is considered. For constant noncarbonate buffer concentrations, DeltaSID = DeltaCB = DeltaVSSB, whereas these equalities no longer hold under changes in noncarbonate buffer concentration. The equivalence is restored if the reference state is changed to include the new buffer concentrations.     

Ultrastructural characteristics of intercellular contacts and bile canaliculi in neonatal rat hepatocytes in primary culture

The ultrastructure of the cellular contacts and bile canaliculi was examined in cultured neonatal (day 5) rat hepatocytes to elucidate the development of cellular polarity. A new scanning electron microscopic technique for cultured hepatocytes allowed a view of cell-cell attachment and the entire cell surface, including the underside on plastic dishes. At 3 h after plating, neonatal hepatocytes were shown to be round, with loss of the preferential localization of cell organelles. After 6 h of culture, the cells had become oblong; they were aggregated in groups of several cells and the cellular contacts were not as rigid or as straight as those in adult hepatocytes. Transmission electron microscopy showed the biliary functional polarity to be like that in vivo. On the undersurfaces of adjacent neonatal hepatocytes a hemicanalicular structure lined with microvilli was found, which probably corresponds to the ultrastructure of bile canaliculi in vivo. However, no canaliculi or orifices of bile channels were found in adult hepatocytes. These results suggest that in neonatal rat hepatocytes the formation of tight rigid cellular contacts was suppressed. Modulation of cell membranes appeared on the undersurfaces of neonatal hepatocytes in early culture stages. The differences in the development of cellular polarity could be caused by the proliferating activity of neonatal hepatocytes.     

Evaluation of the genotoxicity data on caffeine

The potential health effects of caffeine have been investigated for over two decades in a variety of model systems including limited human populations. Thus, it is probably one of the most extensively studied natural occurring dietary chemicals. One area which has received a great deal of attention is the potential genotoxic property of caffeine. To better understand whether caffeine itself or in combination with other agents exhibits genotoxic effects, hundreds of research studies published over the past 5 years have been reviewed. These studies have utilized a number of animal, prokaryotic, eukaryotic, and mammalian cell culture model systems. They have investigated the effects of caffeine alone or in combination with other physical and chemical agents on many aspects of cell division, chromosome stability, toxicity, and mutagenicity. A number of effects have been observed. However, they usually appear after very high doses (> 1 mM) of caffeine in combination with genotoxins, and are usually specific to certain cell types and/or cellular parameters. Humans, on the other hand, consume much less caffeine in the diet, with peak serum levels in the micromolar range 10- and 1000-fold higher compared to levels in animal and cell culture models. Thus, it is difficult to implicate caffeine, even at the highest levels of dietary consumption, as a genotoxin to humans.