Co-cultures of hepatocytes with epithelial-like cell lines: Expression of drug-biotransformation activities by hepatocytes

To improve long-term expression of drug biotransformation activities in hepatocytes, we have examined the suitability of several epithelial-like cell lines (MDCK, MS and L-132) for supporting functional co-cultures with rat hepatocytes. Cells were selected on the basis of their compatibility with hepatocytes, formation of stable monolayers in the absence of serum and lack of drug biotransformation activities. The expression of individual elements of the biotransformation system was evaluated in these co-cultures. Co-cultured hepatocytes remained viable and showed a characteristic polygonal shape for more than a week. Depending on the cell line used, levels of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase activities of co-cultured hepatocytes oscillated between 24–47% of their initial value after 4 days in culture. The highest levels of monooxygenase activity were found in hepatocytes co-cultured with MS cells (41–47%). In contrast, these activities decreased to 6% when hepatocytes were maintained in pure culture for the same period. The activities of the conjugating enzymes UDP-glucuronyltransferase and glutathione S-transferase were maintained at nearly the initial levels during the complete period of study, both in pure and mixed-cultures, regardless of the cell line used. MS cells adapted themselves much better to serum-free culture conditions, and the co-culture with rat hepatocyte was technically easier. After one week, total cytochrome P450 and reduced glutathione in rat hepatocytes/MS co-cultures were 31% and 127% respectively of the day O values, whereas they were undetectable in pure culture. A clear induction of monooxygenase activities by methylcholanthrene, phenobarbital and ethanol could be observed by the 5th day in MS cells/hepatocyte co-cultures. The fact that the results of our work show the suitability of MS cells, an epithelial-derived cell line, for improving the expression of biotransformation enzymes of cultured hepatocytes opens new possibilities of simplifying co-cultures for their use in drug-metabolism studies.Abbreviations AHH aryl hydrocarbon hydroxylase - CDNB 1-chloro-2,4-dinitrobenzene - DMEM Dulbecco's modified Eagle's medium - ECOD 7-ethoxycoumarin O-deethylase - EDTA ethylenediamine tetraacetic acid - Et-OH ethanol - GSH reduced glutathione - GSH-t glutathione S-transferase - MC 3-methylcholanthrene - PB phenobarbital - UDP-Gt UDP-glucuronyltransferase     

Perifusion of co-cultured hepatocytes: optimization of studies on drug metabolism and cytotoxicity in vitro

The combination of co-cultivation of hepatocytes and epithelial cell lines with a newly developed perifusion system was used for in vitro studies on drug metabolism and cytotoxicity. This approach improved the viability and enhanced the induction of the biotransforming capacity of the hepatocytes. As demonstrated for the induction of 7-ethoxyresorufin O-deethylase activity by 3-methylcholanthrene or benzanthracene, co-cultured hepatocytes in the perifusion system responded more sensitively to these inducers than without perifusion, most likely owing to stable (steady-state) concentrations of the inducers under the former conditions and rapidly declining concentrations under the latter conditions. The perifusion approach rendered it possible to determine the kinetics of drug metabolism during single or sequential incubations. After induction with 3-methylcholanthrene and phenobarbital, phase I metabolism of lonazolac to the monohydroxylated product in perifused co-cultures closely (87%) approached the values reported for the in vivo production, whereas in stationary co-cultures only 52% could be reached. Likewise, cytotoxic effects could be detected more precisely in the perifused co-cultures. If cells were pretreated with 0.2 mmol/L galactosamine for 3 h, perifusion with increasing concentrations of menadione differentially killed epithelial RL-ET-14 cells and hepatocytes at low and high concentrations, respectively, while in stationary co-cultures no differential effect was observed and only the higher concentrations were cytotoxic for both cells. Prevention by incubation with S-adenosylmethionine of menadione cytotoxicity up to a menadione concentration of 250 mol/L was seen only in the perifused co-cultures, whereas in stationary cultures only a slight shift of the cytotoxic concentration exerting 50% cell damage to higher values was noted. These results demonstrate the versatile application of perifused co-cultures for studies on drug metabolism including induction of cytochrome P450-dependent enzymes and steady-state kinetics of biotransformation, as well as cytotoxic and protective effects of different drugs.Abbreviations BA benzanthracene - CC₅₀ values cytotoxic concentration exerting 50% cell damage - EROD 7-ethoxyresorufin O-deethylase - LDH lactate dehydrogenase - LON lonazolac - MC 3-methylcholanthrene - PB phenobarbital     

Cytochrome P450 activities in pure and co-cultured rat hepatocytes. Effects of model inducers

Summary The stability and inducibility of several P450 activities (namely, P450 1A1, 2A1, 2B1/2, 2C11, and 3A1) were studied in rat hepatocytes co-cultured with the MS epithelial cell line derived from monkey kidney. The results revealed that these monooxygenase activities were systematically higher in co-cultures than in conventional hepatocyte cultures. Pure cultures showed a rapid loss of monooxygenase activities, which were undetectable after 5 days. In contrast, all isozymes assayed were measurable in co-cultured hepatocytes on Day 7 (about 15 to 40% of the initial activities of Day 0 of culture). The beneficial effects of the co-culture system seemed to be more selective for certain cytochrome P450 isoforms, with P450 1A1 and 3A1 being the best stabilized isozymes after 1 wk. A clear response to inducers was observed in co-cultures, each isozyme showing a different induction pattern. 3-Methylcholanthrene produced a strong increase in P450 1A1 (7-ethoxyresorufin O-deethylase) activity and a low increase in P450 2A1 (testosterone 7α-hydroxylation), whereas no changes were observed in the other activities. Phenobarbital treatment resulted in increases in P450 2B1/2 (7-pentoxyresorufin O-depentylase and 16α- and 16β-hydroxylation of testosterone) activities, while minor effects were observed on P450 3A1 (testosterone 6β-hydroxylation) activity. Dexamethasone markedly increased P450 3A1 (testosterone 6β- and 15β-hydroxylation) activity and, to a lesser extent, P450 2B1/2 (16β-hydroxylation).     

Dependency of the in vitro stabilization of differentiated functions in liver parenchymal cells on the type of cell line used for co-culture

Summary The differentiation status in cultures of primary rat liver parenchymal cells was determined by measuring the activities of various xenobiotic metabolizing enzymes. Most enzyme activities dropped rather rapidly in monocultures of parenchymal cells. The protein content and the activities of cytosolic epoxide hydrolase, glutathione S-transferase, andα-naphthol UDP-glucuronosyl transferase were, however, well stabilized in 7-day-old co-cultures of parenchymal cells with two different lines of rat liver nonparenchymal epithelial cells (NEC1 and NEC2). Phenol sulfotransferase and microsomal epoxide hydrolase activity were reduced in this coculture system after 7 days to about 30 and 20% of the initial activity. Generally, higher enzyme activities were measured in co-cultures with one specific epithelial cell line (NEC2) as compared to those with the other line (NEC1). C3H 10T1/2 mouse embryo fibroblasts supported the parenchymal cells even better than the two epithelial lines, because the activity of microsomal epoxide hydrolase was also stabilized. Glutathione transferase activity was increased over time in this co-culture system. Our results show that the differentiation status of liver parenchymal cells was much better stabilized in co-cultures than in monocultures but that, depending on the type of cells used for co-culture, great quantitative differences existed. The entire pattern of xenobiotic metabolizing enzyme activities could not be stabilized at the kind of levels found in freshly isolated parenchymal cells.     

Effects of L-proline on phase I and phase II xenobiotic biotransformation capacities of rat and human hepatocytes in long-term collagen gel cultures

L-Proline supplementation of the medium for collagen gel cultures of hepatocytes has been shown to improve albumin secretion. A study was made as to whether L-proline is also essential for the maintenance of xenobiotic biotransformation capacities in collagen gel sandwich and immobilisation cultures of rat and human hepatocytes. Key phase I (cytochrome P450-dependent monooxygenase [CYP)] and microsomal epoxide hydrase [mEH]) and phase II (glutathione S-transferase [GST]) biotransformation enzyme activities and the secretion of albumin in the culture medium were assessed in the absence and presence of L-proline. CYP and mEH activities were not affected by the addition of L-proline, whereas phase II alpha-Class GST activity of rat hepatocytes in collagen cultures was decreased. Species differences were demonstrated, as human hepatocytes showed a better maintenance of GST activities than their rat counterparts in the presence of L-proline. Albumin secretion, often considered to be a marker for differentiated cell function, does not parallel the biotransformation capacities of the hepatocytes in culture. Additional results demonstrated an L-proline-mediated enhancement of the proliferation rate of contaminating stellate cells in conventional monolayer culture. Transdifferentiation of stellate cells to proliferating myofibroblasts, along with an increased albumin secretion and collagen synthesis, are characteristic of fibrotic liver. Since the last two phenomena have been observed in L-proline-supplemented collagen gel cultures, it can be concluded that when stable collagen gel cultures of rat hepatocytes are needed for long-term pharmacotoxicological studies, it is preferable to use an L-proline-free culture medium. Further studies on medium optimisation are required for hepatocytes from species other than rat.     

Toxicity of the heterocyclic amine batracylin: investigation of rodent N-acetyltransferase activity and potential contribution of cytochrome P450 3A

The heterocyclic amine, batracylin (BAT), is genotoxic and several lines of evidence suggest that acetylation is one step in the formation of a DNA-damaging product. The variation in susceptibility to BAT toxicity observed between rats and mice has also been linked to the acetylated product. BAT N-acetyltransferase (NAT) activity was determined in rat and mouse hepatic cytosols. Formation of acetylbatracylin (ABAT) was 6 times greater in F-344 hepatic samples compared to either mouse strain, while hepatic BAT NAT activities were similar in C57B1/6 and A/J mice. No deacetylation of ABAT was detected. In contrast, 2-aminofluorene NAT activity in C57B1/6 hepatic cytosol was twice that of the A/J strain and activities in both strains of mice were greater than in rat. Deacetylation of 2-acetylaminofluorene was detected in both species with enzyme activities in C57B1/6>A/J>F-344. Hepatocytes from the F-344 rats, the species most sensitive to BAT toxicity, were used to investigate the contribution of other biotransformation reactions to BAT cytotoxicity. Leakage of cellular lactate dehydrogenase was greater in hepatocytes from male rats than from females, increased on in vivo exposure to dexamethasone, and decreased in the presence of troleandomycin, suggesting that CYP3A-mediated biotransformation of BAT is involved in the formation of a cytotoxic product. When phenol red, a substrate for UDP-glucuronsyltransferase (UDPGT), was absent from the medium, BAT cytotoxicity was reduced. These data are consistent with a role for NAT, CYP, and UDPGT in the biotransformation of BAT. This revised version was published online in July 2006 with corrections to the Cover Date.     

Induction of CYP3A and associated terfenadineN-dealkylation in rat hepatocytes cocultured with 3T3 cells

Long-term culture of hepatocytes has been challenged by the loss of differentiated functions. In particular, there is a rapid decline in cytochrome P450 (CYP). In this study, we cocultured rat hepatocytes with 3T3 fibroblasts for 10 days, and examined hepatocyte viability, morphology, and expression of CYP3A. Terfenadine was incubated with the cultures, and its biotransformation was quantitatively analyzed by HPLC. Terfenadine is metabolized by two major pathways:C-hydroxylation to an alcohol metabolite which is further oxidized to a carboxylic acid, andN-dealkylation to azacyclonol. In rat liver, only theN-dealkylation pathway appears to be mediated by CYP3A since anti-rat CYP3A antibody inhibited azacyclonol but not alcohol metabolite formation in incubations of terfenadine with liver microsomes. Freshly isolated rat hepatocytes were seeded on top of confluent 3T3 cells. Cultures were maintained in Williams' E medium supplemented with 10% fetal bovine serum and either 0.1 mol/L or 5 mol/L dexamethasone. In pure hepatocyte cultures, viability, as determined by lactate dehydrogenase (LDH) activity, decreased steadily to less than 30% of initial levels by day 10. In cocultures, LDH activity remained high and was 70% of initial levels on day 10. The half-life of terfenadine disappearance was optimally maintained in cocultures treated with 5 mol/L dexamethasone, and was associated with the increased formation of azacyclonol. On day 5, nearly 50% of added 5 mol/L terfenadine was converted to azacyclonol within 6 h, whereas the conversion was only 4% on day 1. Western and RNA-slot blot analyses confirmed that treatment with 5 mol/L dexamethasone induced CYP3A mRNA expression and CYP3A protein expression. This coculture system could offer a useful approach in the study of drugs and xenobiotics metabolized by CYP3A.Abbreviations BSA bovine serum albumin - CYP cytochrome P450 - DMSO dimethyl sulfoxide - LDH lactate dehydrogenase - PCN pregnenolone-16-carbonitrile - SDS sodium dodecyl sulfate - SSC saline sodium citrate     

Hepatic biotransformation in the buzzard (Buteo buteo) and the Japanese quail (Coturnix coturnix): effect of PCBs

Hepatic biotransformation was studied in microsomal (cytochrome P-450-dependent monooxygenase activities) and cytosolic (glutathione S-transferase activities) fractions from Japanese quail (Coturnix coturnix) and buzzard (Buteo buteo). Monooxygenase activities were not very different apart from a high 7-ethoxycoumarin de-ethylase activity in quail as compared to buzzard. Glutathione S-transferase activities were higher in quail than in buzzard. DP5 (a commercial mixture of PCBs containing 50% chlorine) produced a marked increase in monooxygenase activity from quail liver. In contrast, no induction was found in buzzard under the same conditions. Glutathione S-transferase activities were not modified in both species.     

Characterization of a primary hepatocyte culture system for toxicological studies

Summary An hepatocyte culture system was developed for potential use in toxicological studiesin vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox™ dishes in a modified Chee’s medium containing 1μM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system forin vitro toxicological studies. A patent has been filed for the use of the system described herein as anin vitro test kit.     

Differential stability of drug-metabolizing enzyme activities in primary rat hepatocytes, cultured in the absence or presence of dexamethasone

The effects of primary hepatocyte culture on the rat cytochrome P450-dependent monooxygenase system and several conjugating enzyme activities were examined using a culture system similar to those used for evaluation of chemicals as potential genotoxins. Cytochrome P450 and cytochrome b5 contents progressively decreased throughout the 72-h culture period to less than 25% of initial values, whereas cytochrome P450 reductase rapidly decreased by 50% during attachment, but then remained stable. Cytochrome P450-dependent testosterone hydroxylase activities decreased more rapidly in culture than did cytochrome P450 content reaching less than 50% of attachment levels by 24 h. Cytochrome P450IIIA immunoreactive protein decreased at a similar rate to testosterone-6 beta-hydroxylase. Activated UDP-glucuronyltransferase activities towards 1-naphthol and testosterone declined more slowly over the 72 h than cytochrome P450 and remained at 50-60% of initial values at 72 h. UDP-glucuronyltransferase activity towards digitoxigenin monodigitoxoside (DIG) did not decrease during culture. Glutathione-S-transferase and sulfotransferase activities also declined during the 72 h at rates which appeared to be isozyme-dependent. Addition of 1 microM dexamethasone (DEX) to the culture medium increased UDP-glucuronyltransferase activity towards DIG, cytochrome P450 reductase and testosterone-6 beta-hydroxylase activities up to 2.5-, 2.0- and 7-fold, respectively and induced cytochrome P450IIIA immunoreactive protein(s) in the hepatocytes after 24 and 48 h of culture; DEX was less effective at the 72 h time-point. DEX treatment also significantly accelerated the decreases in glutathione-S-transferase activities and in sulfotransferase activities towards 1-naphthol and estrone. Thus, it appears that primary rat hepatocytes cultured under standard conditions, not only rapidly lose their monooxygenase capabilities, but also some of their capacity for conjugation. Furthermore, the use of DEX in cell culture medium to enhance cell survival does not maintain total drug-metabolizing enzyme capability, but appears to transiently and selectively increase expression of certain isozymes at the expense of others.     

Intralobular localization of different cytochrome P-450 form dependent monooxygenase activities in the liver of normal and inducer-treated rats.

1. Isolated periportal (PP) and perivenous (PV) hepatocytes from normal and inducer-treated rat livers were used to examine the following: intralobular localization of cytochrome P-450IA, P-450IIB, P-450IIE and P-450IIIA dependent monooxygenase activities and effects of phenobarbital (PB), beta-naphthoflavone (BNF) and pregnenolone-16 alpha-carbonitrile (PCN) on the zonal induction of these monooxygenases. 2. 7-Ethoxyresorufin O-deethylase (7EROD), 7-pentoxyresorufin O-dealkylase (7PROD) and N-nitrosodimethylamine N-demethylase (NAND) activities of PP hepatocytes were not significantly different from those of PV hepatocytes. 3. Ethylmorphine N-demethylase (EMND) activity was significantly higher in PV hepatocytes than in PP hepatocytes of normal rats. 4. EMND activity was induced by PCN and PB treatments. The response of EMND activity to PCN treatment was higher in PP hepatocytes than that in PV hepatocytes, and as a result the PV dominance disappeared following PCN treatment. 5. Extents of the response of this activity to PB treatment were similar in PP and PV hepatocytes, and PV dominance remained unchanged even after induction.     

Sampling and storage conditions of rainbow trout liver affects monooxygenase and conjugation enzymes

1. The effect of storage conditions of rainbow trout (Salmo gairdneri) liver on monooxygenase and conjugation enzyme activities was studied. Fish livers or whole fish were frozen and stored for various periods of time at -4, -20 or -80 degrees C. 2. Freezing the whole fish at -20 degrees C affected the biotransformation enzyme activities dramatically. The loss of monooxygenase activity exceeded up to one-tenth of the initial rate in 17 days. UDP-Glucuronosyltransferase activity increased 50%. Glutathione S-transferase appeared to be the most durable enzyme. 3. When the whole fish were stored in an ice-bath at -4 degrees C for up to 24 hr the activities measured decreased only half of that when frozen for 3 days. 4. When it is impossible to freeze the tissues studied in liquid nitrogen the activities are best preserved when whole, decapitated, bled fish are kept in an ice-bath for less than 24 hr.     

Isolation and culture of hepatocytes from the cynomolgus monkey (Macaca fascicularis)

Summary Isolation and culture techniques for hepatocytes from whole livers of the cynomolgus monkey,Macaca fascicularis, are described. Hepatocytes were isolated by two-step perfusion of livers, using collagenase with hyaluronidase; fructose and trypsin inhibitor were included to reduce cell loss. Yields from a single liver average 4×10⁹ cells with viabilities of 90.8±5.7%. Cells, plated on collagen substrates, were assessed for changes in morphology and various marker enzyme activities over a period of 7 d in culture. Cells exhibited a morphology similar to that observed for this species in vivo; little change in attached and spread cells was observed over the length of time monitored. Enzyme activities for catalase, succinate dehydrogenase, and tyrosine aminotransferase were observed to decrease significantly (though considerable activity remained), whereas acid phosphatase and 5′-nucleotide phosphodiesterase remained unchange. Activity of cytochrome P-450 reductase was observed to increase slightly for the first 2 d, then decrease to about 60% of initial levels. Activity of α-mannosidase was stable for 4 d but was observed to be increased at Day 7. Cells were observed to retain metabolic responsiveness demonstrated by glucose production by both gluconeogenesis and glycogenolysis in response to glucagon stimulation. The monkey hepatocytes obtained by methods described here thus retain hepatocellular morphology and activity through at least 1 wk in culture without medium or culture modification.     

Retention of structural and functional polarity in cultured skate hepatocytes undergoing in vitro morphogenesis

The present study characterized a primary culture model of hepatocytes isolated from the little skate, Leucoraja erinacea, that maintain remarkable structural and functional polarity over 7 days in culture. Skate hepatocytes were isolated as clusters of 3-20 hepatocytes surrounding a bile canaliculus, rather than as single cells. Trypan blue and propidium iodide exclusion was found to be >98%, and the cells maintained high intracellular concentrations of K+, ATP, and reduced glutathione (GSH), and high ratios of ATP/ADP and GSH/GSSG. Glutathione S-transferase activity remained constant, whereas cytochrome P450 activity declined to 16% of initial levels after 7 days. Quantitative RT-PCR analysis revealed that the mRNA levels of several genes remained constant over the 7-day period, whereas Bsep, the canalicular bile salt export pump, levels declined slowly to 30% of initial values. In the presence of dexamethasone, the cells underwent a morphogenesis in which the clusters reannealed into a three-dimensional network of chords. During this morphogenesis, skate hepatocytes clusters maintained a polarized distribution of actin filaments and microtubules, as well as apical and basolateral membrane domains. Polarity of membrane transport systems was confirmed both morphologically, using antibodies raised against Bsep and Mrp2, the canalicular multispecific organic anion transporter, and functionally, by monitoring secretion of the fluorescent organic anions NBD-taurocholate, a Bsep substrate, and fluorescein-methotrexate, an Mrp2 substrate, into the bile canalicular spaces. Overall, the results indicate that in contrast with mammalian hepatocytes, isolated skate hepatocyte clusters retain polarity in culture, and provide an excellent system for investigating long-term effects of drugs and xenobiotics on hepatobiliary functions, and for studying in vitro morphogenesis.     

Oxidative biotransformations by microorganisms: production of chiral synthons by cyclopentanone monooxygenase fromPseudomonas sp. NCIMB 9872

Cyclopentanone monooxygenase, an NADPH- plus FAD-dependent enzyme induced by the growth ofPseudomonas sp. NCIMB 9872 on cyclopentanol, has been utilised as a biocatalyst in Baeyer-Villiger oxidations. Washed whole-cell preparations of the microorganism oxidised 3-hexylcyclopentanone in a regio- but not enantioselective manner to give predominantly the racemic γ-hexyl valerolactone. similar preparations biotransformed 5-hexylcyclopent-2-enone exclusively by regio- plus enantioselective oxidation to the equivalent , β-unsaturated (S)-(+)-δ-hexyl valerolactone (ee = 78%), with no reductive biotransformations catalysed by either EC 1.1.x.x- or EC 1.3.x.x-type dehydrogenases.

An equivalent biotransformation of 5-hexylcyclopent-2-enone was catalysed by highly-purified NADPH- plus FAD-dependent cyclopentanone monooxygenase from the bacterium. The regio- plus enantioselective biotransformation by the pure enzyme of 2-(2′-acetoxyethyl)cyclohexanone yielded optically-enriched (S)-(+ )-7-(2′-acetoxyethyl)-2-oxepanone (ee = 72%). The same biotransformation when scaled up again provided optically-enriched (S)-(+)-ε-caprolactone which was converted, using methoxide, to (S)-(−)-methyl 6,8-dihydroxyoctanoate (ee = 42%). thereby providing a two-step access from the substituted cyclohexanone to this important chiron for the subsequent synthesis of (R-(+)-lipoic acid.

Some characteristics of pure NADPH- plus FAD-dependent cyclopentanone monooxygenase were determined including the molecular weight of the monomeric subunit (50000) of this homotetrameric enzyme, and the N-terminal amino acid sequence up to residue 29, which includes a putative flavin nucleotide-binding site.     

Modulation of functional activities in cultured rat hepatocytes

Rat hepatocytes isolated by enzymatic dissociation of the liver must attach in order to survive for more than a few hours. In conventional culture conditions, they rapidly lose their highly differentiated functions, e.g. adult isozymic forms, enzyme response to specific hormones and cytochrome P-450-dependent monooxygenase activities. Incompletely differentiated cells such as perinatal and regenerating hepatocytes, can transiently exhibit a more differentiated state. Therefore, regulation of hepatic functions, particularly enzyme activities cannot be studied for more than a few days. Hepatocyte survival rate and maintenance of specific functions are dependent on nutrient composition of the medium as well as the substrate. Complex matrices, particularly that derived from the connective liver biomatrix, appear to have an important favorable effect. However, regardless of culture conditions specific functions cannot be quantitatively maintained for more than several days. Recent observations strongly suggest that such a problem may be overcome by mimicking in vivo specific cell-cell interactions. Thus when co-cultured with a liver epithelial cell line, probably derived from biliary ductular cells, adult hepatocytes remain able to synthesize high levels of albumin and to conjugate drugs. In these conditions, the cells secrete an abundant heterogeneous extracellular material. The co-cultures can be maintained in a serum-free medium and specific liver functions can be altered experimentally. Such a model could be appropriate for studying long-term induction and modulation of liver enzyme activities under defined experimental conditions.     

Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions

Specific differentiated gene expression and the morphology of adult rat hepatocytes can be maintained for as long as 8 weeks in vitro only when they are cultured in the presence of biliary epithelial cells; when primary hepatocytes are cultured alone, they lose these functions within 2 to 3 days. We obtained evidence suggesting that contact between hepatocytes and biliary epithelial cells is necessary for maintaining hepatocyte functions. We examined whether junctional communication between and among hepatocytes and biliary epithelial cells is required for long-term maintenance of hepatocyte functions, using a dye-transfer method, in three co-cultures: (1) hepatocytes and biliary epithelial cells prepared from Sprague-Dawley rats; (2) hepatocytes from Sprague-Dawley rats and epithelial cells of the IAR 20 line, originally established from BDVI rats; and (3) hepatocytes from BDVI rats and IAR 20 epithelial cells. The established epithelial cell line (IAR 20) and early-passage cultures of biliary epithelial cells maintained hepatocyte-specific functions in culture for 40 and 70 days, respectively, but the latter induced more stable maintenance of albumin secretion. Hepatocytes cultured alone lost their characteristic morphology within 5 to 8 days, and almost no dye transfer was observed. In co-cultures, the capacity of biliary epithelial cells to communicate among themselves remained relatively high throughout the culture period, whereas hepatocytes showed almost no junctional communication at an early phase of culture and first began to communicate after 2 weeks, communication capacity increasing for at least the next 10 days of culture. The most notable finding was that there was no dye transfer between hepatocytes and biliary epithelial cells in any co-culture system. These results suggest that the maintenance of hepatocyte-specific functions requires intercellular contact but probably not gap-junctional communication between hepatocytes and biliary epithelial cells. This system is useful for studying heterotypic cell-cell interactions and the control of gene expression.     

Pyruvate-induced long-term maintenance of glutathione s-transferase in rat hepatocyte cultures

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione S-transferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, mu and alpha class GST activities were well maintained, whereas GST pi activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: mu,class proteins remained relatively constant, whereas a decrease in the alpha class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, pi class GST activity was increased, but total, mu, and alpha class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.     

Role of cytochrome P450 3A in the metabolism of mefloquine in human and animal hepatocytes

We studied mefloquine metabolism in cells and microsomes isolated from human and animal (monkey, dog, rat) livers. In both hepatocytes and microsomes, mefloquine underwent conversion to two major metabolites, carboxymefloquine and hydroxymefloquine. In human cells and microsomes these metabolites only were formed, as already demonstrated in vivo, while in other species several unidentified metabolites were also detected. After a 48 hr incubation with human and rat hepatocytes, metabolites accounted for 55-65% of the initial drug concentration, whereas in monkey and dog hepatocytes, mefloquine was entirely metabolized after 15 and 39 hrs, respectively. The consumption of mefloquine was less extensive in microsomes, and unchanged drug represented 60% (monkey) to 85-100% (human, dog, rat) of the total radioactivity after 5 hr incubations. The involvement of the cytochrome P450 3A subfamily in mefloquine biotransformation was suggested by several lines of evidence. Firstly, mefloquine metabolism was strongly increased in hepatic microsomes from dexamethasone-pretreated rats, and also in human and rat hepatocytes after prior treatment with a cytochrome P450 3A inducer. Secondly, mefloquine biotransformation in rifampycin-induced human hepatocytes was inhibited in a concentration-dependent manner by the cytochrome P450 3A inhibitor ketoconazole and thirdly, a strong correlation was found between erythromycin-N-demethylase activity (mediated by cytochrome P450 3A) and mefloquine metabolism in human microsomes (r=0.81, P < 0.05, N=13). Collectively, these findings concerning the role of cytochrome P450 3A in mefloquine metabolism may have important in vivo consequences especially with regard to the choice of agents used in multidrug antimalarial regimens.     

Dimethyl sulfoxide enhances lipid synthesis and secretion by long-term cultures of adult rat hepatocytes.

Dimethyl sulfoxide (DMSO) was tested for its effects on lipid metabolism of long-term cultures of adult rat hepatocytes. The addition of 1% DMSO to 3T3-hepatocyte cultures was not toxic to cells and in fact treated cultures maintained better their characteristic morphology for up to 14 days of exposure. DMSO treatment increased 2-3 fold the de novo synthesis of total lipids from[14C]acetate. The analysis by thin layer chromatography of cellular and secreted lipids revealed that DMSO increased the levels of cellular triglycerides, phospholipides and free and sterified cholesterol at 7 days of exposure while at 14 days there was also a 2-3-fold increase in medium secreted lipids. Additionally, DMSO increased the activity of glycerol-phosphate dehydrogenase, a marker enzyme of glycerolipid synthesis, by greater than 50% at either 7 or 14 days of exposure. These results show that 1% DMSO not only is not detrimental to cultured hepatocytes but also enhances lipid synthesis and secretion, both hepatic-differentiated functions.