Cytotoxicity and apoptosis produced by cytochrome P450 2E1 in Hep G2 cells

Two Hep G2 subclones overexpressing CYP2E1 were established with the use of transfection and limited dilution screening techniques. The Hep G2-CI2E1-43 and -47 (E47) cells (transduced Hep G2 subclones that overexpress CYP2E1) grew at a slower rate than parental Hep G2 cells or control subclones that do not express CYP2E1, but remained fully viable. When GSH synthesis was inhibited by treatment with buthionine sulfoximine, GSH levels rapidly declined in E47 cells but not control cells, which is most likely a reflection of CYP2E1-catalyzed formation of reactive oxygen species. Under these conditions of GSH depletion, cytotoxicity and apoptosis were found only with the E47 cells. Low levels of lipid peroxidation were found in the E47 cells, which became more pronounced after GSH depletion. The antioxidants vitamin E, vitamin C, or trolox prevented the lipid peroxidation as well as the cytotoxicity and apoptosis, as did transfection with plasmid containing antisense CYP2E1 or overexpression of Bcl-2. Levels of ATP were lower in E47 cells because of damage to mitochondrial complex I. When GSH was depleted, oxygen uptake was markedly decreased with all substrates in the E47 extracts. Vitamin E completely prevented the decrease in oxygen uptake. Under conditions of CYP2E1 overexpression, two modes of CYP2E1-dependent toxicity can be observed in Hep G2 cells: a slower growth rate when cellular GSH levels are maintained and a loss of cellular viability when cellular GSH levels are depleted. Elevated lipid peroxidation plays an important role in the CYP2E1-dependent toxicity and apoptosis. This direct toxicity of overexpressed CYP2E1 may reflect the ability of this enzyme to generate reactive oxygen species even in the absence of added metabolic substrate.     

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.     

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.     

Characterization of glutathione efflux from Hep G2 cells

Previous studies have suggested that both cAMP-dependent signal transduction pathway and Ca2+/protein kinase C-dependent pathway are involved in GSH efflux from hepatocytes. In the present study, GSH efflux from Hep G2 cells, a human-derived hepatoma cell line, was further characterized. Both epidermal growth factor (0.1-10 ng/ml) and insulin (1 microgram/ml) significantly increased GSH efflux from Hep G2 cells. A fall in the membrane potential produced by the replacement of Na+ with equivalent K+ did not affect GSH efflux significantly. Neither ouabain, a Na+/K+ ATPase inhibitor, vanadate, a Ca2+ ATPase inhibitor, nor BaCl2, a K+ channel blocker, significantly affected the GSH efflux. Methionine (1mM) decreased GSH efflux from the cells, although total GSH content in the cells was not affected during the incubation time of 60 min. Signal transductions through tyrosine kinase-coupled receptors may also be involved in GSH efflux from hepatocytes.     

Intracellular metabolism of human apolipoprotein(a) in stably transfected Hep G2 cells

Lipoprotein(a) [Lp(a)] consists of LDL and the glycoprotein apolipoprotein(a) [apo(a)], which are covalently linked via a single disulfide bridge. The formation of Lp(a) occurs extracellularly, but an intracellular assembly in human liver cells has also been claimed. The human apo(a) gene locus is highly polymorphic due to a variable number of tandemly arranged kringle IV repeats. The size of apo(a) isoforms correlates inversely with Lp(a) plasma concentrations, which is believed to reflect different synthesis rates. To examine this association at the cellular level, we analyzed the subcellular localization and fate of apo(a) in stably transfected HepG2 cells. Our results demonstrate that apo(a) is synthesized as a precursor with a lower molecular mass which is processed into the mature, secreted form. The retention times of the precursor in the ER positively correlated with the sizes of apo(a) isoforms. The mature form was observed intracellularly at low levels and only in the Golgi apparatus. No apo(a) was found to be associated with the plasma membrane. Under temperature-blocking conditions, we did not detect any apo(a)/apoB-100 complexes within cells. This finding was confirmed in HepG2 cells transiently expressing KDEL-tagged apo(a). The precursor and the mature forms of apo(a) were found in the ER and Golgi fractions, respectively, also in human liver tissue. From our data, we conclude that in HepG2 cells the apo(a) precursor, dependent on the apo(a) isoform, is retained in the ER for a prolonged period of time, possibly due to an extensive maturation process of this large protein. The assembly of Lp(a) takes place exclusively extracellularly following the separate secretion of apo(a) and apoB.     

Fatty acid uptake and metabolism in Hep G2 human-hepatoma cells

BACKGROUND: Studies of the mechanisms by which certain water-soluble proteins can assemble into lipid bilayers are relevant to several areas of biology, including the biosynthesis of membrane and secreted proteins, virus membrane fusion and the action of immune proteins such as complement and perforin. The alpha-hemolysin (alpha HL) protein, an exotoxin secreted by Staphylococcus aureus that forms heptameric pores in lipid bilayers, is a useful model for studying membrane protein assembly. In addition, modified alpha HL might be useful as a component of biosensors or in drug delivery. We have therefore used protein engineering to produce variants of alpha HL that contain molecular triggers and switches with which pore-forming activity can be modulated at will. Previously, we showed that the conductance of pores formed by the mutant hemolysin alpha HL-H5, which contains a Zn(II)-binding pentahistidine sequence, is blocked by Zn(II) from either side of the lipid bilayer, suggesting that residues from the pentahistidine sequence line the lumen of the transmembrane channel. RESULTS: Here we show that Zn(II) can arrest the assembly of alpha HL-H5 before pore formation by preventing an impermeable oligomeric prepore from proceeding to the fully assembled state. The prepore is a heptamer. Limited proteolysis shows that, unlike the functional pore, the prepore contains sites near the amino terminus of the polypeptide chain that are exposed to the aqueous phase. Upon removal of the bound Zn(II) with EDTA, pore formation is completed and the sites near the amino terminus become occluded. Conversion of the prepore to the active pore is the rate-determining step in assembly and cannot be reversed by the subsequent addition of excess Zn(II). CONCLUSIONS: The introduction of a simple Zn(II)-binding motif into a pore-forming protein has allowed the isolation of a defined intermediate in assembly. Genetically-engineered switches for trapping and releasing intermediates that are actuated by metal coordination or other chemistries might be generally useful for analyzing the assembly of membrane proteins and other supramolecular structures.     

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.     

Cytotoxicity of aluminum silicates in primary neuronal cultures

To study their cytotoxicity, clays containing aluminum silicates were added to cultures of primary murine spinal cord neurons and differentiated N1E-115 neuroblastoma cells. Bentonite (0.1 mg/ml) and montmorillonite (0.1 mg/ml) rapidly associated with the outer membrane of both N1E-115 and neuronal cells. Erionite (0.1 mg/ml) was randomly distributed throughout the culture. Both bentonite and montmorillonite caused complete cell lysis in the neuronal cultures within 60 min following addition. Erionite had no effect. None of the clays appeared to be cytotoxic to the differentiated N1E-115 cells even though bentonite and montmorillonite were closely associated with the cell membrane. N1E-115 cell lysis did not occur up to 18 h after addition of the clay. Aluminum silicate-containing clays caused a rapid lysis of primary neuronal cells. Differentiated N1E-115 neuroblastoma cells were not susceptible to clay-induced lysis, suggesting that the lytic mechanism is not a general phenomenon that affects all cell types equally.     

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.     

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.     

Metabotropic glutamate receptor agonists stimulate polyphosphoinositide hydrolysis in primary cultures of rat hepatocytes

In phytohemagglutinin (PHA) activated human peripheral blood mononuclear cells. [3H]thymidine uptake and interferon gamma production were increased by the delta-opioid receptor agonist, deltorphin-I (10(-14)-10(-10) M) and by the delta-opioid antagonist naltrindole (10(-13)-10(-9) M). Combination of 10-9 M naltrindole with deltrophin-I (10(-12)-10(-8)M) significantly inhibited the proliferative response but did not affect interferon production.     

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 auramine genotoxicity in primary rat and human hepatocytes and in the intact rat

Auramine, a dye previously found to be a liver carcinogen in both mice and rats, was evaluated for its DNA-damaging and clastogenic activities in primary cultures of rats and human hepatocytes and for the induction of DNA single-strand breaks in the liver and urinary bladder mucosa of intact rats. A similar dose-dependent frequency of DNA fragmentation was revealed by the alkaline elution technique in metabolically competent primary cultures of both rat and human hepatocytes exposed for 20 h to subtoxic concentrations ranging from 10 to 32 microM. In contrast, neither rat nor human hepatocytes displayed an increased frequency of micronuclei after a 48-h exposure to the same auramine concentrations. In rats given a single oral dose of 125, 250 or 500 mg kg-1 auramine, the Comet assay revealed a significant increase in the frequency of DNA lesions in the liver and in the urinary bladder mucosa, the effect being slightly more marked in the liver. Taken as a whole and compared with previous findings, these results suggest that auramine is biotransformed into reactive species in target organs of both rats and humans, and that this dye might play by itself the main role in the increased incidence of bladder cancer which has been judged as causally related to its manufacture.     

Relationship between DNA synthesis and growth factor expression in primary cultures of adult rat hepatocytes

In this study we employed primary culture of adult rat hepatocytes to verify the effects of two different extracellular matrices (collagen, matrigel) on EGF-stimulated DNA synthesis and c-myc expression. Our results confirm that in adult rat hepatocytes EGF induces DNA synthesis, preceded by a transient increase of c-myc expression, when cells are cultured at low density on collagen. DNA synthesis appears to be in reciprocal relationship with hepatic expression of IGF-I, IGFBP-1, IGFBP-2 and IGFBP-4, suggesting that IGF-I/IGFBPs system is not involved in liver growth.     

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.     

Cytotoxicity of polycyclic aromatic hydrocarbon o-quinones in rat and human hepatoma cells

A novel pathway of polycyclic aromatic hydrocarbon (PAH) metabolism involves the oxidation of non-K-region trans-dihydrodiols by dihydrodiol dehydrogenase (DD) to yield PAH o-quinones whose cytotoxicity and genotoxicity are unknown. The cytotoxicity of several PAH o-quinones derived from this reaction [naphthalene-1,2-dione (NPQ), benzo[a]pyrene-7,8-dione (BPQ), and 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBAQ)] was examined in rat (H-4IIe) and human (Hep-G2) hepatoma cells which are known to express DD. 2-Methylnaphthalene-1,4-dione (menadione), a known cytotoxic p-quinone, was used as a positive control. Hepatoma cells (1 x 10(6) cells/mL) were exposed to PAH o-quinones (1-100 microM) for 0-4 h, and cell viability and survival were measured and related to O2.- production and changes in redox potential [GSSG/GSH and NAD(P)+/NAD(P)H]. Three different modes of cytotoxicity were observed: (1) NPQ (no bay region) and DMBAQ (methylated bay region) were as cytotoxic as menadione in reducing cell survival but had less effect on cell viability. These o-quinones adversely affected GSH levels and the redox state of the cell and caused an increase in the production of O2.- in cell suspensions. This cytotoxicity was not enhanced by dicoumarol (10 microM), a DT-diaphorase inhibitor, implying that this enzyme is unable to prevent these PAH o-quinones from entering one-electron redox-cycles. (2) BPQ (bay region only) was the least cytotoxic of the PAH o-quinones studied. BPQ decreased cell viability (< 40% at 20 microM) but did not adversely affect cell survival or the redox state of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of tumor necrosis factor-alpha on glucose and albumin production in primary cultures of rat hepatocytes

Tumor necrosis factor-alpha (TNF) is known to alter significantly in vivo hepatic glucose and albumin metabolism. However, it remains unclear whether the observed effects represent direct actions of this factor or secondary responses due to the recruitment of other mediator systems. The present study was designed to investigate direct actions of TNF on glucose and albumin production in primary cultures of rat hepatocytes. Addition of TNF to the culture medium resulted in a 45% to 50% reduction in glucose production from a control level of 239 +/- 15 nmol/plate.h. This effect was reversed by addition of anti-TNF monoclonal antibody. In glycogen-depleted cells, short-term (5-hour) incubation with TNF did not affect hepatocyte albumin secretion, which was 8.13 +/- 0.29 microgram/plate.h. However, in cells exposed to insulin or in non-glycogen-depleted cells, addition of TNF resulted in a 10% to 25% reduction in albumin production. These findings indicate that TNF exerts direct inhibitory effects on hepatocyte glucose and albumin production, but the effects on the latter process are modest. A notable aspect of the findings is that the albumin effects are insulin or glucose substrate-dependent, which may have implications regarding liver function during nutritional support in critical illness.     

Differential induction of gamma-glutamyl transpeptidase in primary cultures of rat and mouse hepatocytes parallels induction during hepatocarcinogenesis

In carcinogen-treated rats, gamma-glutamyl transpeptidase (GGT) is induced in preneoplastic liver lesions and liver tumors. However, in mice, GGT is rarely detected during hepatocarcinogenesis. Data in this study reveal that GGT is not induced in mouse hepatocytes when they are maintained in vitro under the same conditions that induce GGT activity in primary cultures of rat hepatocytes. GGT activity in rat hepatocytes increased 20-fold during the first 7 days in culture, but there was no induction of GGT in primary cultures of mouse hepatocytes. Comparison of intracellular glutathione levels in rat and mouse liver cells showed that the glutathione level was higher in the mouse liver cells than the rat. Blocking glutathione synthesis with buthionine sulfoximine reduced the intracellular glutathione concentration in mouse liver cells but did not trigger an induction of GGT. Analysis of the GGT mRNA in primary cultures of rat hepatocytes showed that only GGT mRNA(III) is induced. This is the same GGT mRNA species present in preneoplastic hepatic lesions and liver tumors in the rat (1-3). Therefore activation of promoter III in the GGT gene is responsible for induction of GGT in both hepatocytes in vitro and liver tumors in vivo. These data show that primary cultures of rat and mouse hepatocytes provide a model system with which to study interspecies differences in the regulation of this enzyme and to better understand the role of GGT in normal and neoplastic processes.