Mechanism of toxicity of precocene II in rat hepatocyte cultures

Precocene II was more toxic in 24 hour cultures than in 72 hour cultures of rat hepatocytes. In 24 hour cultures, there was no observable toxicity at 75 microM precocene II after exposure for 6 hours, but after 24 hours, 65% of the cells were dead. In contrast, although 794 microM killed 50% of the cells in the 72 hour cultures after a 24 hour exposure, 1 mM killed 96% of the cells within 6 hours. In both 24 and 72 hour cultures, cell death was preceded by a rapid, early loss of mitochondrial membrane potential, followed by decreases in glutathione, reduced pyridine nucleotide status, and plasma membrane Na+/K+-ATPase activity. There was also a rapid loss of ATP in the 72 hour cultures but not in the 24 hour cultures; therefore, onset of cell death may be closely linked to loss of ATP. Inhibition of cytochrome P-450 prevented the toxicity, and partially protected against the loss of membrane potential and glutathione, in 24 hour cultures but was ineffective in 72 hour cultures. Therefore, in addition to depletion of glutathione, precocene II appears to damage mitochondria and plasma membrane functions and can do so by more than one pathway.     

Hepatotoxic effects of SH-reagents in human and rat hepatocyte cultures and in situ perfused rat livers

BACKGROUND: Intravascular ultrasound (IVUS) permits quantitative assessment of the lumen diameter and area of coronary arteries. The experimental study was performed to evaluate the accuracy of diameter and area measurements. METHODS AND RESULTS: Lumen quantitation (lumen diameter D and cross-sectional area A) in lucite tubes (lumen diameter 2.5 to 5.7 mm, Plexiglas) was performed using a mechanical IVUS system (HP console, 3.5 F catheter, Boston Scientific, 30 MHz). The influence of fluid type (blood, water and saline solution), fluid temperature (20 degrees C/37 degrees C), catheter to catheter variation, gain setting and ultrasound frequency (12, 20 and 30 MHz) was determined. In blood at 20 degrees C there was a constant deviation of the measured diameter from the true luminal diameter of -0.29 +/- -0.04 mm (p < 0.06). In water and saline solution at 20 degrees C the mean deviation from true diameter was -0.21 +/- -0.06 mm (p < 0.06). At 37 degrees C, the deviation in blood was greater than at 20 degrees (-0.34 +/- -0.02 mm) which is > 10% in a 3 mm tube (p < 0.06). Three of the ten catheters tested in water at 20 degrees C underestimated true diameter by more than -0.3 mm. The deviation from true diameter (5 mm tube) with varying gain settings was -0.14 mm to -0.23 mm compared to -0.19 mm at standard settings (p > 0.288). At 12 MHz diameter measured was over-estimated. The error in absolute area estimation increased with increasing diameter tested in blood at 37 degrees C (-1.21 to -2.72 mm2), whereas the relative error ([Measured Area-True Area]/True Area x 100 [%]) was more striking at smaller diameters (up to -25% in the 2.5 mm tube). CONCLUSION: Luminal diameters and areas are underestimated by this particular IVUS system. When IVUS imaging and measurements are made during coronary interventions this error should be taken into account with regard to appropriate sizing of the device and the assessment of the postprocedure result. Because systematic errors might also occur in other IVUS system (not tested in this study), it is advisable to ensure that each system is validated prior to clinical use, especially when exact measurements are required.     

Effects of dithiocarbamates on toxicity of cadmium in rat primary hepatocyte cultures

The protective effects of N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD) on the toxicity of Cd in the rat primary hepatocyte cultures were studied. Cytotoxicity was assessed by measuring cell viability, extra cellular lactic dehydrogenase (LDH) activity, and intracellular lipid peroxidation and active oxygen species. Primary hepatocyte cultures were treated with 109CdCl2 (5, 10 or 50 microM Cd and 1.7 KBq of 109Cd/well) for 30 min or 4 h. BGD or HBGD was added to the culture medium to make the final concentration of 100 microM and incubated for 4.5 h in 30 min Cd exposure or 1 h in 4 h Cd exposure. Decreases in the hepatocyte viability caused by all Cd exposure concentrations were significantly prevented by treatment with BGD or HBGD. The treatment with the chelating agents for 4.5 h after Cd exposure for 30 min significantly prevented increases in extracellular LDH activity. Increases in the lipid peroxidation in hepatocytes exposed to Cd for 30 min or 4 h were prevented significantly by treatment with BGD or HBGD for 4.5 h or 1 h, respectively. Moreover, the increases in the level of active oxygen species caused by Cd exposure for 30 min were significantly prevented by treatment with the chelating agents for 1.5 h. These findings suggest that BGD and HBGD protect against the cytotoxicity of Cd in rat primary hepatocyte cultures and that the protective effects of chelating agents presumably result from a decrease in the Cd level, the effective sequestration of the reactive Cd ion, and the direct preventive effect on the active oxygen species in the hepatocytes.     

Enhanced oxygen delivery reverses anaerobic metabolic states in prolonged sandwich rat hepatocyte culture

It must be assumed that current petri dish primary hepatocyte culture models do not supply sufficient amounts of oxygen and thus cause anaerobic metabolism of the cells. This is contrary to the physiologic state of the cells. In vivo the liver is a highly vascularized organ with a rather high blood flow rate of a mixture of arterial and venous blood. The aim of the present study was to show the oxygen dependence of primary rat hepatocytes in long-term culture and to define appropriate conditions that could allow hepatocytes to maintain tissue specific functions in an aerobic environment. To this purpose matrix overlaid hepatocytes were either cultured on gas-permeable (fluorinated hydrocarbon films) or gas-impermeable (polystyrene) supports at 10% and 20% ambient oxygen concentration (v/v), respectively. Tissue-specific functions were assessed by studying albumin and urea secretion as well as xenobiotic metabolism. The mRNA expression and catalytic activities of the cytoprotective antioxidant enzymes mitochondrial manganese superoxide dismutase (MnSOD), cytosolic copper and zinc superoxide dismutase, peroxisomal catalase, and cytosolic glutathione peroxidase were investigated to assess intracellular responses to the defined variations in oxygen supply. Hepatocytes could successfully be maintained at aerobic conditions in long-term culture on gas-permeable PTFE films. At 50% (10%, v/v) of currently used oxygen levels lactate accumulation was prevented, a plateau-like albumin secretion reestablished, urea secretion improved, and xenobiotic metabolism proceeded at physiological rates. mRNA expression of cytoprotective enzymes responded to the pericellular availability of oxygen and was most pronounced in the case of MnSOD. However, the biggest stress factor for the hepatocytes still appeared to be the isolation procedure, as mRNA expression and catalytic activities were most elevated shortly thereafter. In conclusion, this study clearly shows the oxygen dependence of primary rat hepatocytes in long-term culture and indicates means to establish appropriate conditions for the aerobic culture of primary rat sandwich hepatocytes with full maintenance of function. The long-term culture of hepatocytes on oxygenating supports at in vivo-like oxygen tensions therefore appears to be more physiologic and beneficial for the cells.     

Hepatobiliary effects of tertiary-butylhydroperoxide (tBOOH) in isolated rat hepatocyte couplets

CD40 ligand (CD40L) is involved in the T-cell-dependent regulation of B-cell growth and survival and can rescue normal germinal centre B cells and several types of malignant B cells from apoptosis in vitro. We have previously reported that serum of patients with chronic lymphocytic leukaemia contained elevated levels of biologically active soluble CD40L (sCD40L). Whether an augmented CD40L pathway exists in patients with other types of B-cell lymphoid malignancies and the source of native sCD40L in these patients is currently unknown. Using a sensitive ELISA assay, soluble CD40L (sCD40L) was detected in the sera of both healthy individuals and patients with haematological malignancies; however, its level was significantly elevated only in patients with B-cell lymphomas (P<0.0001). Several types of malignant B cells coexpressed CD40 and CD40L proteins, and CD40L mRNA was detected in purified resting malignant B cells. The dual expression of CD40 and CD40L in B cells and the presence of native sCD40L in human serum suggest that a direct T-B-cell contact may not be required for CD40L delivery to B cells. This data raises the possibility that an autocrine cytokine loop involving CD40L may contribute to the growth regulation of benign and malignant B cells in vivo.     

Drug metabolism in hepatocyte sandwich cultures of rats and humans

Adult hepatocytes from rat and man were maintained for 2 weeks between two gel layers in a sandwich configuration to study the influence of this culture technique on the preservation of basal activities of xenobiotic-metabolizing phase I and phase II enzymes. The response of these enzyme activities to an enzyme inducer was investigated using rifampicin (RIF). Basal levels of cytochrome P-450 (CYP) isozymes were characterized by measuring ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and the specific oxidation of testosterone (T). In hepatocytes from untreated rats, CYP isozyme levels, including the major form CYP 2C11, increased during the first 3 days in culture. After this period of recovery, the levels of CYP 2C11, CYP 2A1, and CYP 2B1 decreased, whereas CYP 3A1 increased. In contrast to these dynamic changes, CYP activities such as CYP 1A2 and the major isozyme CYP 3A4 were largely preserved until day 9 in cultures of human hepatocytes. In measuring phase II activities, a distinct increase in glucuronosyltransferase (UDP-GT) activity toward p-nitrophenol (PNP) was found for rat and human hepatocytes over 2 weeks in culture. Sulfotransferase (ST) activity toward PNP showed an initial increase, with a maximum at day 7 and day 9 in culture, respectively, and then decreased until day 14. Glutathione S-transferase (GST) activity decreased constantly during the time of culture. Effects of the enzyme-inducing drug rifampicin on phase I and phase II enzymes were investigated using cultured human hepatocytes. Rifampicin treatment (50 micromol/L) for 7 days resulted in a 3.7-fold induction of CYP 3A4 at day 9 in culture. ECOD activity was increased sixfold and phase II ST activity increased twofold compared to the initial value at day 3. No effect of rifampicin on CYP 3A was found in cultures of rat hepatocytes. These results demonstrate that rat and human hepatocytes preserve the major forms of CYP isozymes and phase II activities and respond to inducing drugs such as rifampicin. The novel hepatocyte sandwich culture is suitable for investigating drug metabolism, drug-drug interactions and enzyme induction.     

Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.     

Bioartificial liver support: developments in hepatocyte culture and bioreactor design

Bioartificial liver devices aim to support patients with acute liver failure (ALF) until orthotopic liver transplantation (OLT) or spontaneous recovery due to hepatic regeneration can occur. However, initial clinical experiences with two devices have indicated that functional efficacy in this setting may be less than in the experimental situation. Several fundamental issues remain unresolved, including the cell mass required to provide meaningful support and which of those hepatocyte components and bioreactor designs so far proposed is best able to do this. In particular, further studies of the efficacy of devices incorporating human hepatocyte lines transformed by either cultural conditions or genetic engineering and those based on multi-channel or flat bed bioreactor designs in which hepatocytes are co-cultured with non-parenchymal cells are awaited. Controlled trials on a multicentre basis in well-defined patient groups and with standardised outcome measures will be required to properly evaluate the clinical value of these devices. A better understanding of factors promoting recovery and regeneration of the native liver and to what extent these can be provided by extracorporeal devices will be essential to the further development of effective bioartificial liver support systems.     

Macrophage-induced inhibition of nitric oxide production in primary rat hepatocyte cultures via prostaglandin E2 release

Kupffer cells and other macrophages play an important role in pathogenesis of toxicants in the liver. The aim of this study was to evaluate the effect of macrophages on hepatocyte production of nitric oxide (NO), which has been previously reported to be protective toward oxidative stress induced in primary rat hepatocytes. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocytes at various ratios between macrophages and hepatocytes. These cocultures were supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) for 23 hours to induce NO synthase and trigger NO production. NO production was followed by quantification of nitrites in culture medium and dinitrosyl iron complexes (DNIC) in intact hepatocytes after separation from macrophages. In cocultured hepatocytes incubated with LPS and IFN-gamma, DNIC and nitrite levels decreased compared with those observed in hepatocytes cultured without macrophages in the same conditions. Moreover, inhibition of NO production in hepatocyte cocultures was macrophage-number-dependent. Macrophage-conditioned medium also inhibited NO production in hepatocytes, suggesting that the effect of macrophages was mediated by soluble factors. Among the soluble factors known to decrease NO levels are some cytokines, growth factors, reactive oxygen species, and prostaglandins. Ultrafiltration of macrophage-conditioned medium through a 500-d membrane to rule out higher-molecular-weight molecules, such as anti-inflammatory cytokines and growth factors, failed to restore NO production. In the same way, the use of superoxide dismutase (SOD) and catalase (CAT) to eliminate reactive oxygen species produced by macrophages did not lead to recovery of NO levels in hepatocytes. However, when NO synthesis was inhibited in macrophages by NG-monomethyl-L-arginine (L-NMMA), hepatocytes recovered the capacity to produce NO. A net decrease of prostaglandin E2 (PGE2) release by macrophages was concomitantly observed. Moreover, inhibition of PGE2 production in macrophages by indomethacin led to restoration of NO levels. Taken together, our observations suggest that NO synthesized by macrophages can decrease NO production in hepatocytes via PGE2 release. Because of the protective role of NO toward many liver injuries, it may be postulated that macrophages contribute through this mechanism to liver damage.     

Ameloblast-lineage cells of rat tooth germs proliferate and scatter in response to hepatocyte growth factor in culture

Hepatocyte growth factor (HGF) is considered to be one of the mediators of epithelial-mesenchymal interactions during early organogenesis and to be involved in the development of murine molars. In this study, the immunohistochemical localization of HGF and of its receptor, c-Met, revealed that HGF was distributed in the proliferating mesenchymal cells in the dental papillae and that c-Met was continuously expressed in the epithelial cells during the development of rat incisors. These observations confirmed the involvement of HGF in the development of rat incisors, as demonstrated previously in molars. We then used a primary culture of ameloblast-lineage cells, prepared from mandibular incisors of young rats, to examine the direct effects of HGF on the growth and differentiation of ameloblasts. We found that HGF at 2-20 ng/ml induced a marked increase in the number of ameloblast-lineage cells and in the scattering of such cells. Our results suggest that HGF promotes the proliferation and scattering of ameloblast-lineage cells simultaneously.     

Inhibition of sphingolipid biosynthesis in rat primary hepatocyte cultures by fumonisin B1 and other structurally related compounds

Plaque cholesterol is thought to be derived exclusively from low-density lipoproteins that have become trapped and modified in the subendothelial space of arterial vessels. However, in this study we provide the first visual evidence that demonstrates that (a) remnants of post-prandial lipoproteins rapidly penetrate arterial tissue; (b) efflux is not complete; and (c) focal accumulation of chylomicron remnants occurs within the subendothelial space. The arterial retention of chylomicron remnants is consistent with atherogenesis being in part a post-prandial phenomenon.     

Effect of time in culture on modulation of Na(+)-K(+)-ATPase activity in rat alveolar type II cells

To determine the effect of time in culture on epithelial cell function, we evaluated the modulation of Na(+)-K(+)-ATPase activity in rat alveolar type II cells in culture. Ouabain sensitivity testing revealed that the alpha-1 predominance in the enzyme's isoforms was maintained over the 120 hours in culture. Basal Na(+)-K(+)-ATPase activity in the whole cell homogenate did not differ significantly between cells cultured for 48 hours and those cultured for 120 hours. Terbutaline (10 mM) did not activate Na(+)-K(+)-ATPase in the cells cultured for 48 hours, but, it significantly increased the activity of this enzyme in the cells cultured for 120 hours cells cultured for 48 hours, produced intracellular cyclic AMP after exposure to 10 mM of terbutaline. These results indicate that the coupling between Na(+)-K(+)-ATPase and the beta-adrenergic pathway in alveolar type II cells can be influenced by the time in cell culture.     

Pleiotypic response of rat heart cells in culture to serum stimulation

The pleiotypic effects of medium replacement were studied in rat heart cell cultures. After each medium change alpha-aminoisobutyric acid and glucose transport are increased, RNA and protein syntheses are activated. DNA synthesis did not begin before 12 hours and was followed by a wave of mitoses. This sequence of events suggests that the stimulated cells were in early G 1 phase. DNA synthesis, following the shift to a fresh medium, is linearly related to the amount of serum used as is protein synthesis. However when serum concentrations higher than 20 percent were used no increased protein synthesis could be observed suggesting the existence of another limiting factor, which was probably the isoleucine content of the medium. The serum stimulating factor is heat stable, dialysable and was found in both human and fetal calf sera.     

DNA repair synthesis induced by azo dyes in primary rat hepatocyte cultures using the bromodeoxyuridine density-shift method

Ante- and post-mortem bloodstains prepared from the blood of volunteers and corpses were analysed for ATP and its related compounds by reversed-phase high-performance liquid chromatography (HPLC). The results showed that (1) ATP was present in a large amount in antemortem bloodstains but not in postmortem stains, (2) AMP, adenosine, inosine, hypoxanthine, xanthine and uracil either were not detected or were detected in smaller amounts in antemortem than in postmortem bloodstains, and (3) ADP was present in both ante- and post-mortem bloodstains. These differences suggest that quantitation of these compounds may be useful in identifying whether bloodstains are ante- or post-mortem.     

Biological spin trapping. II. Toxicity of nitrone spin traps: dose-ranging in the rat

To obtain the strongest possible free radical spin adduct signal using the electron paramagnetic resonance spectroscopy-spin trapping technique, it is desirable to load an animal with the highest dose of spin trap possible. One hundred and twenty six male Sprague-Dawley rats were used to establish the toxic dose range for PBN (alpha-phenyl N-tert butyl nitrone) and 18 other similar spin traps. The lethal dose of PBN was found to be approximately 100 mg/100 g BW (0.564 mmol/100 g. The 18 other compounds were then tested, and their toxicities were gauged in terms of molar equivalents to PBN. Of these spin traps, DMPO (5,5-dimethyl-1-pyrroline-N-oxide) was found to be the least toxic (no toxic signs at twice the lethal dose for PBN) while 2,6-difluoro-PBN and M4PO (3,3,5,5-tetramethyl-1-pyrroline-N-oxide) were the most toxic, both causing death at one eighth the PBN-equivalent lethal dose. Nine of the 18 nitrones appeared non-toxic at the 0.25 PBN-equivalent lethal dose level.     

Parenting in different cultures: time to focus

This commentary proposes that cross-cultural studies of parenting, like studies within a culture, should become more focused--with antecedent or concurrent measures selected to provide a clear view toward "child outcome." Furthermore, differences within countries may be as great as differences between countries. Therefore, the relevant cultural context, which itself may be changing, needs careful specification.     

Dynamics of time discrimination: II. The effects of multiple impulses

According to a diffusion generalization model, time discrimination is determined by the frequency and recency of preceding intervals of time. A procedure for studying rapid timing was used to investigate whether pigeons' wait-time responses were sensitive to these factors. In Experiment 1 the number (two or eight) and spacing (consecutive or far apart) of 5-s interfood intervals (called impulses) intercalated in a series of 15-s interfood intervals (nonimpulses) were studied. Experiment 2 was identical to the first but the interfood intervals were increased by a factor of three. Overall, impulses shortened wait times in the next interfood interval. However, several impulses occurring in succession extended the localized effect of an impulse: Wait times following a set of eight-close impulses were slow to recover to preimpulse levels. The results show that linear waiting is only an approximation to the dynamic process, and a process that is sensitive to events in an animal's remote past, such as the diffusion generalization model, provides a better account of rapid timing effects.