Hepatobiliary function and toxicity in vitro using isolated hepatocyte couplets

• 1.1. Hepatocyte couplets can be routinely prepared from rat liver to produce a suitable in vitro model for polarized primary cells.
• 2.2. Centrifugal elutriation provides a means of producing enriched subpopulations of periportal and perivenous couplets from the same liver, thus providing a means of studying the influence of zonal heterogeneity on hepatobiliary function.
• 3.3. The maintenance of structural and secretory polarity demonstrated by hepatocyte couplets provides a convenient in vitro system for mechanistic studies of factors both regulatory and adversely affecting hepatobiliary functions.
• 4.4. Couplets are also uniquely appropriate for specific studies of regulation at the biliary pole, on the performance of junctions and on the maintenance and rate of transcytotic movement.
• 5.5. The possibility also exists that effects of an in vivo pre-exposure to agents causing hepatobiliary dysfunction can be assessed in couplets ex vivo.

     

Ethionine toxicity in vitro: the correlation of data from rat hepatocyte suspensions and monolayers with in vivo observations

The hepato-steatogenic compound ethionine has been used to investigate the correlations between in␣vivo and in vitro toxicity data. The aim was to find a suitable model of toxicity in hepatocyte suspensions or monolayers in vitro, which could predict the known toxicity of ethionine in vivo and which could be implemented in screening compounds of unknown toxicity. Thus a variety of markers of cytotoxicity, metabolic competence and liver-specific functions were investigated in rat hepatocyte suspensions and monolayers and compared with in vivo data in the rat. The following markers were measured in the appropriate system: (1) Neutral red uptake; 3-(4,5 dimethyl)thiazol-2-yl,-2,5-diphenyl tetrazolium bromide (MTT) reduction; lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) leakage (cytotoxicity). (2) ATP levels, protein synthesis and glutathione (GSH) levels (metabolic competence). (3) Urea and triglyceride synthesis and β-oxidation (liver specific functions). Ethionine (0–30 mM) did not affect the markers of direct cytotoxicity, except neutral red uptake, which was reduced by 18 and 30 mM ethionine after 20 h in culture. ATP and GSH depletion occurred in hepatocyte suspensions at the highest concentrations of ethionine (20 and 30 mM) after 1 h. In monolayers, GSH levels were reduced after 4 h, but not 20 h. Urea synthesis was increased in hepatocyte suspensions from 1 to 3 h by 10–30 mM ethionine and reduced after 20 h in cultured hepatocytes (18–30 mM). Protein synthesis was reduced and β-oxidation was increased in ethionine-treated hepatocyte suspensions. Unfortunately, there was no measurable effect on triglyceride accumulation within cells (the major biochemical change in␣vivo) in either system. Ethionine treated hepatocytes in suspension showed the same rate of triglyceride synthesis and transportation out of cells as control cells. Thus, hepatocyte suspensions were able to mimic the early biochemical effects of ethionine in vivo (ATP and GSH depletion, inhibition of protein synthesis) and some effects on urea synthesis, but monolayer cultures appeared to be less sensitive to the toxicity of ethionine. However, neither in vitro system was able to model the effects of ethionine on the accumulation of triglycerides in vivo. Received: 16 June 1998 / Accepted: 29 June 1998     

Paracetamol metabolism and toxicity in isolated hepatocytes from rat and mouse

Hepatocytes isolated from mouse and rat catalyzed the formation of glucuronide, sulphate, glutathione and cysteine conjugates of paracetamol. These metabolites were separated by high pressure liquid chromatography. 1. Sulphation had higher affinity for paracetamol than glucuronidation in hepatocytes from both mouse and rat, whereas glucuronidation had higher capacity. The maximal rate of glucuronidation was similar in hepatocytes from both species, the rate of sulphation was, however, several-fold less in hepatocytes from mouse. 2. Formation of the glutathione conjugate was directly correlated with loss of intracellular glutathione (GSH). The rate of glutathione conjugate formation increased about three times in rat hepatocytes after phenobarbital treatment. This induced rate was, however, only half of that in hepatocytes from control mouse. In both species the reaction was saturated only at very high paracetamol concentrations. The rate of formation of the cysteine conjugate was very low compared to the other reactions. 3. Only hepatocytes isolated from mouse lost integrity, measured as increased permeability of the cell membranes, upon incubation in the presence of paracetamol.     

Investigations on the in vitro metabolism of five synthetic 19-norprogestins using hepatocyte suspensions isolated from five laboratory animal species

Five synthetic progestins of the 19-nortestosterone type (norethisterone, NET; levonorgestrel, LN; gestodene, GEST; NET-3-oxime, NETO; norgestimate, NGM) were investigated in the in vitro hepatocyte model. Radiolabelled progestins were added to hepatocyte suspensions (3 x 10(6) cells/ml) freshly prepared from female rat, guinea pig, rabbit, dog (beagle) and cynomolgus monkey. Drug level decreases (NET, LN, GEST) and prodrug conversions (NETO, NGM) were followed by radiochromatography (HPLC) for 60 min. In the case of NET and NETO the conversion into ethinyl estradiol (EE2) was quantified by RIA after HPLC separation. Half-lives of drug level decreases (t1/2), areas under the curves (AUC) and metabolic clearance rates (MCR) were estimated for all progestins. For NETO and NGM the percentages of conversion into NET and LN were calculated, respectively, and levels of EE2 determined in the case of NET and NETO. Rat hepatocytes showed an extremely high metabolic activity towards NET, LN and GEST resulting in t1/2 values of below 2 min. Respective values for rabbit hepatocytes ranged from 5-8 min, whereas half-lives calculated for liver cells from guinea pig, dog and monkey were generally above 30 min. A drastic increase in t1/2 was found for NETO (as compared to NET) in hepatocytes from rat, rabbit and monkey but not from guinea pig. Dog hepatocytes degraded NETO about 3 times more rapidly than NET. NGM was degraded much faster than LN in hepatocytes from all species except the rat. Liver cells from guinea pig and dog seem to be able to metabolize the 3-oxime group much more rapidly than hepatocytes from the other animal species. The lowest degree of prodrug conversion of 4% was observed for NGM and dog hepatocytes. Elevated EE2 levels were found in all experiments with NET and NETO. Results of NET, LN and GEST were compared with published in vivo experiments. No correlations were found for t1/2, MCR, and AUC.     

Metabolic activity of fresh and cryopreserved dog hepatocyte suspensions

1. Dog hepatocytes were cryopreserved at 6 × 10⁶ viable cells/ml in a suspension buffer containing 10% DMSO and were stored in liquid nitrogen. 2. The exclusion of trypan blue dye was 96± 2 and 85± 9% in fresh and cryopreserved (CP) hepatocytes, respectively. Albumin synthesis was unaffected by freezing. 3. Ethoxycoumarin and ethoxyresorufin O-deethylase activities were equivalent in fresh and CP hepatocytes. 4. The profile of testosterone metabolism was unaffected by freezing. Total hydroxylase activities were 815± 33 pmol/min/10⁶ CP cells in freshly isolated whole hepatocytes and 463± 24 pmol/min/10⁶ CP whole hepatocytes, but they were equivalent in fresh and CP hepatocyte homogenates supplemented with 250 μM NADPH. 5. Phase 2 enzymes were functional in freshly thawed CP hepatocytes but they required exogenous addition of cofactors (20 μM UDPGA and 1.7 μM PAPS). 6. When placed in suspension for longer times, fresh and CP cell viabilities were 88± 6 and 64± 2% after 4 h. ECOD and EROD activities were equivalent in fresh and CP hepatocyte suspensions, over 4 h. Testosterone hydroxylase activities were well maintained in fresh cell suspensions but they declined to 63± 6% of the initial activity after 4 h in CP hepatocytes. 7. These results indicate that CP dog hepatocytes are a suitable in vitro system for xenobiotic metabolisms in ce enzyme functions in CP hepatocytes were stabilized. Cofactors in freshly thawed CP hepatocytes should be measured and controlled for optimal use.     

Toxicity and biotransformation of volatile halogenated anesthetics in rat hepatocyte suspensions

This study examines the toxicity (as measured by reduced intracellular K+) and metabolism (defluorination) of halothane and enflurane in rat hepatocytes in suspension (RHS) with regards to O2 tension, time, and concentration. In 95% O2 halothane is more toxic than enflurane when RHS are exposed to 5-20 microliters of these anesthetics. At these levels halothane is not metabolized while enflurane is metabolized. At 21% O2 a similar pattern was seen with regards to toxicity. However, metabolism of halothane rapidly reached an elevated level while that of enflurane is reduced when compared to 95% O2. Thus toxicity of halothane and enflurane at these dose levels appears to be unrelated to metabolism and due solely to a solvent effect.     

In vitro zonation and toxicity in a hepatocyte bioreactor.

The complex architecture of the liver is intertwined with its response to xenobiotic compounds. In particular, hepatocyte subpopulations are distributed along the sinusoid in zones 1 to 3, leading to prototypical "periportal" and "centrilobular" patterns of cell death in response to a toxic insult. In vitro models that more closely represent these zones of sub-specialization may therefore be valuable for the investigation of hepatic physiology and pathophysiology. We have established a perfused hepatocyte bioreactor that imposes physiologic oxygen gradients on co-cultures of rat hepatocytes and non-parenchymal cells, thereby producing an in vitro model of zonation. In order to predict and control oxygen gradients, oxygen transport in a parallel-plate bioreactor containing co-cultures was first mathematically modeled and experimentally validated. Co-cultures exposed to these physiologic oxygen gradients demonstrated regionally heterogeneity of CYP2B and CYP3A protein that mimics the distribution seen in the zonated liver. The distribution of CYP expression in the bioreactor was shown to vary with exposure to different chemical inducers and growth factors, providing a potential platform to study physiologic zonal responses. In order to explore zonal hepatotoxicity, bioreactors were perfused with APAP (acetominophen) for 24 h, resulting in maximal cell death at the low-oxygen outlet region similar to centrilobular necrotic patterns observed in vivo. This hepatocyte bioreactor system enables further in vitro investigation into zonation-dependent phenomena involving drug metabolism and toxicity.     

Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

l-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 °C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-l-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher l-methionine-l-sulfoxide levels relative to control hepatocytes, whereas the increases in l-methionine-d-sulfoxide (Met-d-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-d-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-dl-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.     

Effect of binding protein surface charge on palmitate uptake by hepatocyte suspensions

1. Studies were directed at determining whether hepatocytes, isolated from female Sprague-Dawley rats, facilitate the uptake of protein-bound long-chain fatty acids. We postulated one form of facilitated uptake may occur through an ionic interaction between the protein-ligand complex and the cell surface. These interactions are expected to supply additional ligand to the cell for uptake.
2. The clearance rate of [³H]-palmitate in the presence of α₁-acid-glycoprotein (pI=2.7), albumin (pI=4.9) and lysozyme (pI=11.0) was investigated. Palmitate uptake was determined in the presence of protein concentrations that resulted in similar unbound ligand fractions (=0.03). The experimental clearance rates were compared to the theoretical predictions based upon the diffusion-reaction model.
3. By use of our experimentally determined equilibrium binding and dissociation rate constants for the various protein-palmitate complexes, the diffusion-reaction model predicted clearance rates were 4.9 μl s⁻¹/10⁶ cells, 4.8 μl s⁻¹/10⁶ cells and 5.5 μl s⁻¹/10⁶ cells for α₁-acid-glycoprotein, albumin and lysozyme, respectively; whereas the measured hepatocyte palmitate clearance rates were 1.2±0.1 μl s⁻¹/10⁶ cells, 2.3±0.3 μl s⁻¹/10⁶ cells and 7.1±0.7 μl s⁻¹/10⁶, respectively.
4. Hepatocyte palmitate clearance was significantly faster (P<0.01) in the presence of lysozyme than albumin which was significantly faster than α₁-acid-glycoprotein (P<0.01). The marked difference in clearance rates could not be explained by considering differences in solution viscosity.
5. Our results are consistent with the notion that ionic interactions between protein-ligand complexes and the cell surface facilitate the ligand uptake by decreasing the diffusional distance of the unbound ligand and/or by facilitating the protein-ligand dissociation rate.

     

Acrylamide toxicity in isolated rat hepatocytes

Acrylamide (ACR) is an important industrial chemical used primarily in the production of polymers and co-polymers. Acrylamide is mainly neurotoxic to experimental animals as well as humans and has also been shown to be mutagenic and carcinogenic. The present study was designed to investigate the toxicity of ACR on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and were incubated with different concentrations of ACR (0.1, 1, 10 m ) for 2 hours. Cell viability by trypan blue exclusion and leakage of the enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) were determined. Reduced glutathione (GSH), glutathione S-transferase (GST) activity were also measured. A significant decrease in the cell viability was observed after exposure to 10 m ACR for 30 min, while 1 m ACR caused a significant decrease in the viability after 60 min. ALT leakage was parallel to the cell viability. AST leakage was significantly increased at 30 min of incubation with 10 m ACR, whereas 2 hours of incubation was required for the leakage of AST from rats hepatocytes with 1 m ACR. 10 m ACR decreased significantly GSH as early as 30 min, while GSH level was decreased at 60 min after exposure to 1 m ACR. Also, the GST activity increased with increasing the dose of ACR. Cytochrome P450 concentration was decreased after exposure to 10 m ACR. The effect of ACR on cell viability, ALT and AST leakage, GSH and GST activity was time and dose dependent.     

Copper toxicity in isolated rat hepatocytes

In relative excess, copper is a cytotoxic metal. The injury may be related to the process of lipid peroxidation. Isolated hepatocytes provide a suitable system for an examination of this aspect of copper toxicity. Furthermore, interactions between copper and agents that protect against its toxic effects in vivo can be examined at a cellular level by use of isolated hepatocytes. Therefore, isolated rat hepatocytes were incubated with varying cupric chloride concentrations (5–200 μm) for up to 90 min. The copper caused a concentration and time-related decrease in cell viability as assessed by loss of intracellular potassium ion (K⁺) and aspartate aminotransferase (AST). An increase in lipid peroxidation and a decrease in reduced glutathione were also observed in response to copper. Of several potentially interactant compounds tested, only chromic chloride, diethyldithiocarbamate, and penicillamine were found to reduce the loss of K⁺. Ammonium molybdate alone and in combination with sodium sulfate were able to markedly decrease the release of AST from the hepatocytes. While the antioxidants, butylated hydroxyanisole and N,N′-diphenyl-p-phenylenediamine decreased the lipid peroxidation attributable to copper, they had no protective effects against loss of cell viability. This suggests that lipid peroxidation is not the cause of the injurious effects of this metal in isolated rat hepatocytes.     

Furosemide antagonizes the contractile response to noradrenaline on isolated rat tail artery

To determine the effect of furosemide (FUR) on the noradrenaline (NA) contraction of arteries, experiments were performed on isolated rat tail arteries according to Nicholas. FUR (300 mumol/l, 3 mmol/l) as well as Ca2+ transport system inhibitor, verapamil (VER), decreased the NA contractile force and FUR (3 mmol/l) antagonized the effect of VER (10 and 100 nmol/l). Calculated dose ratios according to Stephenson indicated that FUR and VER compete not only with NA but with each other. FUR decreased Ca2+ concentration in rat arteries studied in vivo by atomic absorption spectrophotometric method as well. It could be assumed that FUR depresses the sensitivity to NA and antagonizes NA induced contraction of arteries acting on transmembrane Ca2+ transport system.     

Effects of culture duration on hydrogen peroxide-induced hepatocyte toxicity

The effects of culture duration on primary cultured mouse hepatocyte antioxidant levels (superoxide dismutase, catalase, glutathione peroxidase, vitamin E, and glutathione) and susceptibility to glucose oxidase (GO)- and hydrogen peroxide (H2O2)-induced cell killing and lipid peroxidation were examined. Membrane fatty acid composition was also evaluated. Adult male B6C3F1/CrlBR mouse hepatocytes were isolated by collagenase perfusion of the liver and cultured on 60-mm plastic dishes in Leibovitz's L-15 medium supplemented with glucose (1 mg/ml), dexamethasone (1 microM), fetal bovine serum (10%, v/v), and gentamicin sulfate (50 micrograms/ml) for 0 hr (freshly isolated cells) to 96 hr. Hepatocyte toxicity (determined by lactate dehydrogenase release and lipid peroxidation) after a 2-hr exposure to GO (0.8-80 micrograms/ml) or H2O2 (1-5 mM) decreased with increased time in culture. This decreased hepatocyte sensitivity to GO and H2O2 toxicity was not related to antioxidant enzyme activity since superoxide dismutase, catalase, and glutathione peroxidase declined during the 96-hr culture period. In contrast, glutathione and vitamin E levels in the cultured hepatocytes rose to 274.9 +/- 8.3% and 220.6 +/- 18.6% of the levels in freshly isolated cells (129.6 +/- 11.5 nmol and 0.10 +/- 0.01 nmol per 10(6) hepatocytes, respectively). The percentage of polyunsaturated fatty acids in hepatocyte phospholipids and triglycerides decreased with culture duration while the percentage of oleic acid increased in esterified and free fatty acid pools after 2 hr in culture. Total fatty acids were not affected by time in culture. These results suggest that the decreased hepatocyte susceptibility to the toxic effects of hydrogen peroxide may have been due to elevations in cellular GSH and vitamin E levels and decreases in membrane polyunsaturated fatty acids. The data also indicate that hepatocytes in primary culture undergo changes in antioxidant levels and fatty acid composition that may affect free radical toxicity at different times in culture.     

Chlordecone-induced follicular toxicity in mouse ovaries

The effect of the pesticide, chlordecone, on murine follicular development was examined. Female CD-1 mice were exposed to chlordecone for 5 consecutive days for each of 4 consecutive weeks (0.25 mg/day). Controls received sesame oil vehicle or estradiol-17 beta (E-17 beta; 0.1 mg/day) since chlordecone has been ascribed estrogenic activity. Animals were sacrificed 24 h following the final exposure. Ovaries were removed, serially sectioned, and stained. Follicles were classified as small, medium, or large and were tabulated. Twice as many medium-sized follicles were found in the E-17 beta-treated mice as in both the chlordecone-exposed and sesame oil control groups. Both pesticide- and E-17 beta-exposed mice displayed a much higher percent of atresia in the large follicles; however, there were more actual healthy, large follicles in the E-17 beta group. Thus, both chlordecone and E-17 beta induced increased atresia among large follicles, which could be due to the estrogenicity of these agents. However, a decreased pool of healthy large- and medium-sized follicles occurred in chlordecone-treated mice, a condition not seen in E-17 beta-treated mice. Thus, the pool of potentially ovulatory follicles is reduced in the pesticide-treated animals.     

Effect of volatile anesthetics on protein synthesis and secretion by rat hepatocyte suspensions

The effect of volatile anesthetics on protein synthesis and secretion by isolated rat hepatocytes in suspension was investigated. Halothane and enflurane inhibited protein synthesis in a dose-dependent manner. Diethyl ether had little effect on protein synthesis while isoflurane caused a mild inhibition. This effect was more pronounced in hepatocytes from phenobarbital treated male rats when compared to hepatocytes from control rats. Protein synthesis in hepatocytes from phenobarbital treated female rats was inhibited similar to that seen with control male rat hepatocytes. Isoflurane, enflurane, and halothane also caused a dose-dependent inhibition of protein secretion, while diethyl ether was only mildly inhibitory. From these studies it appears that inhibition of protein synthesis and secretion might be an early and sensitive indicator of cellular injury by volatile anesthetics.     

Amphetamine toxicity in isolated and aggregated mice

Summary The phenomenon of increased amphetamine toxicity in aggregated mice was studied by placing one mouse treated with amphetamine among either 9 untreated (1 A+9 U) or 9 sedated (1 A+9 S) mice. Mortalities were recorded at the fifth and twentieth hour following intraperitoneal injection of dl-amphetamine in doses ranging from 15 to 125 mg/kg. The mortality in these two groups were compared with the mortality in two other groups where one amphetaminetreated mouse was isolated (1 A) and where 10 amphetamine-treated mice were grouped together (10 A). The highest mortality occurred when all the mice in the aggregate received amphetamine (10 A), whereas, the lowest mortality occurred in mice (1 A) isolated after amphetamine injection. The mortality curves for mice placed among untreated (A + 9 U) or sedated (1 A + 9 S) mice were similar and lay in between those for the isolated (1 A) and aggregated (10 A) mice. A biphasic pattern was noted in the dose-mortality curves for the three different aggregated groups.Supported in part by the U.S. Public Health Service Research Grant RH 00507-01 National Center for Radiologic Health and the American Cancer Society (Milwaukee).     

Benoxaprofen induced toxicity in isolated rat hepatocytes

The toxicity of benoxaprofen, a non-steroidal anti-inflammatory compound was investigated using rat hepatic microsomal and isolated hepatocyte suspensions. In microsomes, benoxaprofen produced a Type I binding spectra and competitively inhibited (k_i 380 μM) the oxidative metabolism of aminopyrine. Marked toxicity was observed following incubation of benoxaprofen with isolated hepatocytes from either untreated, phenobarbitone (PB) or 3-methylcholanthrene (3-MC) pretreated male rats. In untreated hepatocytes increases in the intracellular lactate/pyruvate (L/P) ratio and alanine aminotransferase (ALT) release were related to the benoxaprofen concentration and duration of incubation. Alterations in L/P ratio preceded the release of cytosolic ALT and at 4 h a well defined dose-response relationship existed between the benoxaprofen concentration and the observed increases in the L/P ratio and ALT release. Pretreatment of animals with either PB or 3-MC did not affect the temporal nature nor the magnitude of the hepatocyte response to benoxaprofen. In addition, inhibitors of cytochrome P-450 isozymes (SKF-525A, metyrapone and -napthoflavone) were ineffective with regard to modifying the observed toxicity. The results of this study suggest that hepatic cytochrome P-450 mediated metabolism may not be implicated in the toxicity of benoxaprofen in isolated hepatocytes. However, alterations in the cellular redox state and evidence of plasma membrane bleb formation suggest that benoxaprofen may uncouple oxidative phosphorylation and disturb intracellular calcium ion homeostasis.