The formaldehyde metabolic detoxification enzyme systems and molecular cytotoxic mechanism in isolated rat hepatocytes

The toxicity and carcinogenicity of formaldehyde (HCHO) has been attributed to its ability to form adducts with DNA and proteins. A marked decrease in mitochondrial membrane potential and inhibition of mitochondrial respiration that was accompanied by reactive oxygen species formation occurred when isolated rat hepatocytes were incubated with low concentrations of HCHO in a dose-dependent manner. Hepatocyte GSH was also depleted by HCHO in a dose-dependent manner. At higher HCHO concentrations, lipid peroxidation ensued followed by cell death. Cytotoxicity studies were conducted in which isolated hepatocytes exposed to HCHO were treated with inhibitors of HCHO metabolising enzymes. There was a marked increase in HCHO cytotoxicity when either alcohol dehydrogenase or aldehyde dehydrogenase was inhibited. Inhibition of GSH-dependent HCHO dehydrogenase activity by prior depletion of GSH markedly increased hepatocyte susceptibility to HCHO. In each case, cytotoxicity was dose-dependent and corresponded with a decrease in hepatocyte HCHO metabolism and increased lipid peroxidation. Antioxidants and iron chelators protected against HCHO cytotoxicity. Cytotoxicity was also prevented, when cyclosporine or carnitine was added to prevent the opening of the mitochondrial permeability transition pore which further suggests that HCHO targets the mitochondria. Thus, HCHO-metabolising gene polymorphisms would be expected to have toxicological consequences on an individual's susceptibility to HCHO toxicity and carcinogenesis.     

Relationships between formaldehyde metabolism and toxicity and glutathione concentrations in isolated rat hepatocytes

The metabolism and toxicity of formaldehyde (CH2O) in isolated rat hepatocytes was found to be dependent upon the intracellular concentration of glutathione (GSH). Using hepatocytes depleted of GSH by treatment with diethyl maleate (DEM), the rate of CH2O (5.0 mM) disappearance was significantly decreased. Formaldehyde decreased the concentration of GSH in hepatocytes, probably by the extrusion of the CH2O-GSH adduct, S-hydroxymethylglutathione. Formaldehyde toxicity was potentiated in cells pretreated with 1.0 mM DEM as measured by the loss of membrane integrity (NADH stimulation of lactate dehydrogenase (LDH) activity) and an increase in lipid peroxidation (formation of thiobarbituric acid-reactive compounds). This potentiation of toxicity was both CH2O concentration-dependent and time-dependent. There was an excellent correlation between the increase in lipid peroxidation and the decrease in cell viability. L-Methionine (1.0 mM) both protected the cells from toxicity caused by the combination of 8.0 mM CH2O and 1.0 mM DEM and increased the cellular GSH concentration. The antioxidants, ascorbate, butylated hydroxytoluene (BHT) and alpha-tocopherol (10, 25 and 125 microM), all exhibited dose-dependent protection against toxicity produced by 8.0 mM CH2O and 1.0 mM DEM. At toxic concentrations of CH2O (10.0-13.0 mM), administered by itself, lipid peroxidation did not increase concomitantly with the decrease in cell viability and the addition of antioxidants (125 microM) did not influence CH2O toxicity. These results suggest that CH2O toxicity in GSH-depleted hepatocytes may be mediated by free radicals as a result of the effect of CH2O on a critical cellular pool of GSH. However, cells with normal concentrations of GSH are damaged by CH2O by a different mechanism.     

Molecular cytotoxic mechanisms of catecholic polychlorinated biphenyl metabolites in isolated rat hepatocytes

Polychlorinated biphenyl (PCB) and PCB metabolites are highly lipophilic and accumulate easily in the lipid bilayer and fat deposits of the body. The molecular cytotoxic mechanisms of these metabolites are still not understood. The aim of the present study was to compare the cytotoxicity and toxicological properties of six dihydroxylated metabolites using isolated rat hepatocytes. All of the metabolites were more cytotoxic than 4-chlorobiphenyl (4-ClBP) and less cytotoxic than phenyl hydroquinone (PHQ). The order of cytotoxic effectiveness of catecholic metabolites expressed as LC(50) (2h) was 3',4'-diCl-2,3-diOH-biphenyl>PHQ>4'-Cl-2,5-diOH-biphenyl, 4'-Cl-2,3-diOH-biphenyl>2',5'-diCl-3,4-diOH-biphenyl>2',3'-diCl-3,4-diOH-biphenyl>3',4'-diCl-3,4-diOH-biphenyl>4'Cl-3,4-diOH-biphenyl>4'-Cl-biphenyl; showing that the positions of hydroxyl and chlorine groups were important for their hepatotoxicity and that the two 2,3-diOH congeners were the most cytotoxic. Cytotoxicity for 3,4-diOH metabolites correlated with the number and position of chlorine atoms with the more chlorine atoms being more cytotoxic. The cytotoxic order of metabolites with two chlorine atoms being 2',5'>2',3'>3',4'. Borneol, an uridine diphosphate glucuronosyltransferases (UGT) inhibitor, increased the cytotoxicity of all tested metabolites; suggesting that glucuronidation was a major mechanism of elimination of these compounds. On the other hand entacapone, a catechol-O-methyl transferase (COMT) inhibitor, only increased the cytotoxicity of 3',4'-diCl-3,4-diOH-biphenyl, 3',4'-diCl-2,3-diOH-biphenyl and 4'-Cl-2,3-diOH-biphenyl. Hepatocyte GSH was depleted (oxidized and conjugated) by these metabolites before cytotoxicity ensued in a similar order of effectiveness to their cytotoxicity with PHQ being the most effective. Hepatocyte mitochondrial membrane potential also decreased before cytotoxicity ensued with a similar order of effectiveness as their cytotoxicity. These results suggest that catecholic cytotoxicity can be attributed to mitochondrial toxicity and oxidative stress. Semiquinone or benzoquinone species were also important in the cytotoxicity of catecholic metabolites.     

The application of microcalorimetry to the assessment of metabolic efficiency in isolated rat hepatocytes

1. Heat output by suspensions of isolated rat hepatocytes was determined by using a modified batch-type microcalorimeter. 2. The ratio of O(2) uptake (determined polarographically) to heat output was used to assess the metabolic efficiency of isolated hepatocytes. 3. Cells from starved or fed rats incubated in either bicarbonate-buffered physiological saline containing gelatin, or bicarbonate-buffered physiological saline containing amino acids, serum albumin and glucose showed no significant difference with respect to the ratio of O(2) uptake to heat output. 4. For liver cells from 24h-starved rats, the addition of 10mm-dihydroxyacetone and 2.5mm-fructose significantly decreased the ratio of O(2) uptake to heat output from 1.94+/-0.05 in the controls to 1.52+/-0.04 and 1.54+/-0.01mumol/J respectively. 5. Glucagon (1mum), which slightly increased both O(2) uptake and heat output, did not significantly alter the ratio. 6. The addition of extracellular 10mm-NH(4)Cl and urease to provide an energetically wasteful cycle by ensuring hydrolysis of newly synthesized urea, lowered the ratio of O(2) uptake to heat output from 1.81+/-0.08 to 1.47+/-0.06mumol/J, indicating a reduced metabolic efficiency. 7. Metabolic efficiency in rats of different dietary regimen, age and genetically based obesity was also assessed. No differences in the ratio of O(2) uptake to heat output were found between liver cell suspensions prepared from rats maintained on colony diet and high-fat diet or sucrose-rich diet nor between animals ranging from 38 to 179 days of age. Comparison of the ratio of liver cell O(2) uptake to heat output between homozygote Zucker fa/fa obese rats and their lean littermates showed no significant difference. 8. It is concluded that the ratio of O(2) uptake to heat output for isolated hepatocytes is relatively constant unless perturbed by conditions that markedly enhance substrate cycling.     

Autofluorescence properties of isolated rat hepatocytes under different metabolic conditions.

The contribution of endogenous fluorophores - such as proteins, bound and free NAD(P)H, flavins, vitamin A, arachidonic acid - to the liver autofluorescence was studied on tissue homogenate extracts and on isolated hepatocytes by means of spectrofluorometric analysis. Autofluorescence spectral analysis was then applied to investigate the response of single living hepatocytes to experimental conditions resembling the various phases of the organ transplantation. The following conditions were considered: 1 h after cells isolation (reference condition); cold hypoxia; rewarming-reoxygenation after cold preservation. The main alterations occurred for NAD(P)H and flavins, the coenzymes strictly involved in energetic metabolism. During cold hypoxia NAD(P)H, mainly the bound form, showed an increase followed by a slow decrease, in agreement with the inability of the respiratory chain to reoxidize the coenzyme, and a subsequent NADH reoxidation through alternative anaerobic metabolic pathways. Both bound/free NAD(P)H and total NAD(P)H/flavin ratio values were altered during cold hypoxia, but approached the reference condition values after rewarming-reoxygenation, indicating the cells capability to restore the basal redox equilibrium. A decrease of arachidonic acid and vitamin A contributions occurred after cold hypoxia: in the former case it may depend on the balance between deacylation and reacylation of fatty acids, in the latter it might be related to the vitamin A antioxidant role. An influence of physico-chemical status and microenvironment on the fluorescence efficiency of these fluorophores cannot be excluded. In general, all the changes observed for cell autofluorescence properties were consistent with the complex metabolic pathways providing for energy supply.     

Metabolic mechanisms of methanol/formaldehyde in isolated rat hepatocytes: carbonyl-metabolizing enzymes versus oxidative stress

Methanol (CH(3)OH), a common industrial solvent, is metabolized to toxic compounds by several enzymatic as well as free radical pathways. Identifying which process best enhances or prevents CH(3)OH-induced cytotoxicity could provide insight into the molecular basis for acute CH(3)OH-induced hepatoxicity. Metabolic pathways studied include those found in 1) an isolated hepatocyte system and 2) cell-free systems. Accelerated Cytotoxicity Mechanism Screening (ACMS) techniques demonstrated that CH(3)OH had little toxicity towards rat hepatocytes in 95% O(2), even at 2M concentration, whereas 50 mM was the estimated LC(50) (2h) in 1% O(2), estimated to be the physiological concentration in the centrilobular region of the liver and also the target region for ethanol toxicity. Cytotoxicity was attributed to increased NADH levels caused by CH(3)OH metabolism, catalyzed by ADH1, resulting in reductive stress, which reduced and released ferrous iron from Ferritin causing oxygen activation. A similar cytotoxic mechanism at 1% O(2) was previous found for ethanol. With 95% O(2), the addition of Fe(II)/H(2)O(2), at non-toxic concentrations were the most effective agents for increasing hepatocyte toxicity induced by 1M CH(3)OH, with a 3-fold increase in cytotoxicity and ROS formation. Iron chelators, desferoxamine, and NADH oxidizers and ATP generators, e.g. fructose, also protected hepatocytes and decreased ROS formation and cytotoxicity. Hepatocyte protein carbonylation induced by formaldehyde (HCHO) formation was also increased about 4-fold, when CH(3)OH was oxidized by the Fenton-like system, Fe(II)/H(2)O(2), and correlated with increased cytotoxicity. In a cell-free bovine serum albumin system, Fe(II)/H(2)O(2) also increased CH(3)OH oxidation as well as HCHO protein carbonylation. Nontoxic ferrous iron and a H(2)O(2) generating system increased HCHO-induced cytotoxicity and hepatocyte protein carbonylation. In addition, HCHO cytotoxicity was markedly increased by ADH1 and ALDH2 inhibitors or GSH-depleted hepatocytes. Increased HCHO concentration levels correlated with increased HCHO-induced protein carbonylation in hepatocytes. These results suggest that CH(3)OH at 1% O(2) involves activation of the Fenton system to form HCHO. However, at higher O(2) levels, radicals generated through Fe(II)/H(2)O(2) can oxidize CH(3)OH/HCHO to form pro-oxidant radicals and lead to increased oxidative stress through protein carbonylation and ROS formation which ultimately causes cell death.     

Thiols in formaldehyde dissimilation and detoxification

Glutathione is not a universal coenzyme for formaldehyde oxidation. MySH (mycothiol, 1-O-(2'-[N-acetyl-L-cysteinyl]amido-2'-deoxy-alpha-D-glucopyranosyl)-D-m yo-inositol) is GSH's counterpart as coenzyme in formaldehyde dehydrogenase from certain gram-positive bacteria. However, formaldehyde dissimilation and detoxification not only proceed via thiol-dependent but also via thiol-independent dehydrogenases. The distinct structures and enzymatic properties of MySH-dependent and GSH-dependent formaldehyde dehydrogenases could provide clues for development of selective drugs against pathogenic Mycobacteria. It is to be expected that other new types of thiol-dependent formaldehyde dehydrogenases will be discovered in the future. Indications exist that the product of thiol-dependent formaldehyde oxidation, the thiol formate ester, is not only hydrolytically converted into thiol and formate but can also be oxidatively converted in some cases by a molybdoprotein aldehyde dehydrogenase into the corresponding carbonate ester, decomposing spontaneously into CO2 and the thiol.     

The assessment of viability in isolated rat hepatocytes.

Isolated rat hepatocytes are used in many metabolic studies, but the viability of these cell preparations is often not adequately established. The present study shows that ATP content is a more reliable index of metabolic viability than trypan blue exclusion. At some of the low trypan blue exclusion levels quoted in the literature, a high percentage of cell preparations is likely to be nonviable by the criterion of ATP content. We suggest that ATP content measured on initial cell preparations and at the end of all incubation procedures is essential for establishing cell viability for metabolic studies on isolated hepatocytes.     

Biotransformation and cytotoxic effects of hydroxychavicol, an intermediate of safrole metabolism, in isolated rat hepatocytes

The biotransformation and cytotoxic effects of hydroxychavicol (HC; 1-allyl-3,4-dihydroxybenzene), which is a catecholic component in piper betel leaf and a major intermediary metabolite of safrole in rats and humans, was studied in freshly isolated rat hepatocytes. The exposure of hepatocytes to HC caused not only concentration (0.25-1.0 mM)- and time (0-3 h)-dependent cell death accompanied by the loss of cellular ATP, adenine nucleotide pools, reduced glutathione, and protein thiols, but also the accumulation of glutathione disulfide and malondialdehyde, indicating lipid peroxidation. At a concentration of 1 mM, the cytotoxic effects of safrole were less than those of HC. The loss of mitochondrial membrane potential and generation of oxygen radical species assayed using 2′,7′-dichlorodihydrofluoresein diacetate (DCFH-DA) in hepatocytes treated with HC were greater than those with safrole. HC at a weakly toxic level (0.25 and/or 0.50 mM) was metabolized to monoglucuronide, monosulfate, and monoglutathione conjugates, which were identified by mass spectra and/or ¹H nuclear magnetic resonance spectra. The amounts of sulfate rather than glucuronide or glutathione conjugate predominantly increased, accompanied by a loss of the parent compound, with time. In hepatocytes pretreated with either diethyl maleate or salicylamide, HC-induced cytotoxicity was enhanced, accompanied by a decrease in the formation of these conjugates and by the inhibition of HC loss. Taken collectively, our results indicate that (a) mitochondria are target organelles for HC, which elicits cytotoxicity through mitochondrial failure related to mitochondrial membrane potential at an early stage and subsequently lipid peroxidation through oxidative stress at a later stage; (b) the onset of cytotoxicity depends on the initial and residual concentrations of HC rather than those of its metabolites; (c) the toxicity of HC is greater than that of safrole, suggesting the participation of a catecholic intermediate in safrole cytotoxicity in rat hepatocytes.     

Sodium-dependent taurocholate uptake by isolated rat hepatocytes occurs through an electrogenic mechanism

The uptake mechanism for the bile salt, taurocholate, by the liver cell is coupled to sodium but the stoichiometry is controversial. A one-to-one coupling ratio would result in electroneutral transport, whereas cotransport of more than one sodium ion with each taurocholate molecule cause an electrogenic response. To better define the uptake of this bile salt, we measured the effect of taurocholate on the membrane potential and resistance of isolated rat hepatocytes using conventional microelectrode electrophysiology. The addition of 20 microM taurocholate caused transient but significant depolarization accompanied by a significant decrease in membrane resistance. The electrical effect induced by taurocholate mimicked that induced by L-alanine (10 mM), the uptake of which is known to occur through an electrogenic, sodium-coupled mechanism. The sodium dependence of taurocholate-induced depolarization was further confirmed by: (1) replacing Na+ with choline +, and (2) preincubating cells with ouabain (2 mM) or with the Na+-ionophore, gramicidin (25 micrograms/ml); both suppressed the electrogenic response. Further, cholic acid, which inhibits sodium-coupled taurocholate uptake in hepatocytes, inhibited taurocholate evoked depolarization. These results support the hypothesis that sodium-coupled taurocholate uptake by isolated hepatocytes occurs through an electrogenic process which transports more than one Na+ with each taurocholate molecule.     

Effects of piperine on enzyme activities and bioenergetic functions in isolated rat liver mitochondria and hepatocytes

The influence of piperine on the enzymes and bioenergetic functions in isolated rat liver mitochondria and hepatocytes was studied. Piperine at lower concentrations (<50 μM) did not affect the RCR and ADP:O ratios, state 4 and 3 respirations supported by site-specific substrates, viz. glutamate + malate, succinate, and ascorbate + TMPD. The site-specific effects became significantly apparent only at higher concentrations. Only the state 3 respiration supported by NAD-linked substrates was impaired equipotently in mitochondria and permeabilized hepatocytes; the effect appeared to be localized at energy-coupling site 1. In hypotonic treated mitochondria, respiration supported by three kinds of substrates was not affected. Among the respiratory chain-linked enzymes, the activity of NADH-dehydrogenase registered a significant decrease of about 25, 42, and 53% at 100, 150, and 180 μM piperine, respectively. The activity of Mg⁺⁺-ATPase, however, was stimulated at concentrations above 150 μM. Among the matrix enzymes, only malate and succinate dehydrogen-ases were studied. Malate dehydrogenase only showed a strong concentration-related inhibition in both the forward and backward directions. Enzyme kinetics indicated noncompetitive inhibition with a very low Ki of 10 μM. The presence of unsaturated double bonds in the side chain of piperine appeared essential for producing this strong inhibition. The studies suggested that piperine produces concentration related site-specific effects on mitochondrial bioenergetics and enzymes of energy metabolism.     

Glucose metabolic alterations in isolated and perfused rat hepatocytes induced by pancreatic cancer conditioned medium: a low molecular weight factor possibly involved

A serious insulin resistance characterizes pancreatic cancer-associated diabetes mellitus. Elsewhere, we demonstrated that MIA PaCa2 cultured cells secrete a soluble factor responsible for reduced glucose tolerance induced in SCID mice. The intracellular mechanism of insulin resistance was investigated in isolated and perfused rat hepatocytes incubated with MIA PaCa2 conditioned medium. Lactate production was reduced compared to hepatocytes incubated with control medium while 1,2-DAG was increased and PKC was activated in the hepatocytes incubated with MIA PaCa2 conditioned medium. This behavior was not reproduced treating the hepatocytes with the growth factors EGF, interleukin Ibeta, interleukin-6, and TGF-beta1. In an attempt to make a biochemical identification of the hypothesized tumor associated-diabetogenic factors we observed a low molecular weight protein in the conditioned medium, absent in the nonconditioned one, that may be responsible for the described behaviors.     

Effect of hypoxic cell radiosensitizers on glutathione level and related enzyme activities in isolated rat hepatocytes

A comparative study of the effect of misonidazole and novel radiosensitizers on glutathione (GSH) levels and related enzyme activities in isolated rat hepatocytes was performed. Incubation of hepatocytes with 5 mM radiosensitizers led to a decrease in the intracellular GSH level. The most pronounced decrease in cellular GSH was evoked by 2,4-dinitroimidazole-1-ethanol (DNIE); after incubation for only 15 min, GSH was hardly detected. DNIE-mediated GSH loss was dependent upon its concentration. DNIE reacted with GSH nonenzymatically as well as with diethylmaleate, while misonidazole and 1-methyl-2-methyl-sulfinyl-5-methoxycarbonylimidazole (KIH-3) did not. Addition of partially purified glutathione S-transferase (GST) did not enhance DNIE-mediated GSH loss in a cell-free system. DNIE inhibited glutathione peroxidase (GSH-Px), GST, and glutathione reductase (GSSG-R) activities in hepatocytes, while misonidazole and KIH-3 did not. GSH-Px activity assayed with H2O2 as substrate was the most inhibited. Inhibition of GSH-Px activity assayed with cumene hydroperoxide as substrate and GST was less than that of GSH-Px assayed with H2O2 as substrate. GSSG-R activity was decreased by DNIE, but not significantly. Incubation of purified GSH-Px with DNIE resulted in a little change in the activity when assayed with H2O2 as substrate.     

Factors influencing sulfation in isolated rat hepatocytes

Sulfation of harmol by isolated hepatocytes was dependent on an exogenous source of sulfate. Inorganic sulfate ion stimulated sulfation by over ten fold. Analysis of the stimulation of harmol sulfation by sulfate indicated a K_m of 239 μM and a V_max of 1.1 μmoles harmol sulfate/min/10⁶ cells. Cysteine also stimulated the rate of harmol sulfation but was less effective than sulfate ion. Lithium chloride inhibited harmol sulfation. Sulfation was unaffected by several metabolic alterations which inhibited harmol glucuronidation. Fasting for 24 hours, and incubation with ethanol or linoleic acid, did not influence the rate of sulfation but inhibited glucuronidation by 50 percent.     

Amino acid transport in isolated rat hepatocytes

Summary Improvements in the collagenase perfusion techniques have made isolated rat hepatocytes a popular model in which to study hepatic function. Our knowledge of hepatic amino acid transport has been advanced as a result of this methodology. Translocation across the hepatocyte plasma membrane can, in some instances, represent the rate-limiting step in the overall metabolism of certain amino acids. Furthermore, regulation of amino acid uptake by hepatocytes appears to play a role in diabetes, and perhaps in malignant transformation. Comparisons between normal adult hepatocytes and several hepatoma cell lines show basic differences in amino acids transport. There are at least eight distinct systems in normal hepatocytes for transport of the amino acids. One of these, System A, transports the small neutral amino acids most efficiently and responds to a wide variety of hormones. Systems A and N exhibit enhanced uptake rates after the cells have been maintained in the absence of extracellular amino acids, a phenomenon termed adaptive control. Further studies using isolated hepatocytes will increase our basic understanding of membrane transport processes and their regulation.     

Metallothionein induction in freshly isolated rat hepatocytes

The control of metallothionein (MT) synthesis was investigated in freshly prepared rat hepatocytes in experiments of short-term duration. Viability and metabolic function were maintained in incubations of 6-h duration. MT synthesis was measurable in hepatocytes from fed rats at Zn concentrations down to 1 μM. Zn and dexamethasone induced concentration-dependent increases in the synthesis of MT with maximal increases above the 5-h control of 3.2- and 2.5-fold, respectively. Zn induction of MT was first measurable at 2 h and was inhibited by actinomycin C. Although initial (0 h) MT concentrations in hepatocytes from fasted rats were double those from fed rats, after 6-h incubation in the presence of 50 μM Zn, the fasted rat hepatocytes showed only half the MT concentrations of the fed rat hepatocytes. Glucagon and interleukin-6 (IL-6) were less effective inducers and increased MT synthesis by 28 and 17%, respectively. IL-6 (100 U/mL) was found to have an additive effect on MT synthesis above that of Zn alone (1–50 μM) or Zn plus dexamethasone (1 μM). A supernatant from LPS-stimulated macrophages increased MT synthesis by 40%. The basal MT synthesis was not increased by either tumor necrosis factor-α (TNF-α) or interleukin-1 (IL-1). All incubations were carried out in the presence of RPMI 1640 medium with Hepes (20 mM), bicarbonate (24 mM), and fatty acid-free albumin (FAFA; 0.5% w/v). MT synthesis was also seen using Krebs bicarbonate buffer with glucose (10 mM), Hepes (20 mM), and FAFA (0.5% w/v), and although the level of MT synthesis was less than in RPMI, the increases in concentrations of MT at 5 h were 225, 139, 36 and 20% for Zn, dexamethasone, glucagon, and control, respectively. It is concluded that MT synthesis occurs in freshly prepared hepatocytes and that these cells are responsive to some of the established inducers of MT. This system enables the study of MT synthesis in individual rats in various metabolic and pathological states.     

Bile acid synthesis in isolated rat hepatocytes

Normal adult rat hepatocytes were incubated for 48h and the concentration of total and individual bile acids in homogenized samples of the culture was measured at intervals during the incubation, using radiogas chromatography and isotope derivative assay. The net increase in bile acids over the value observed at the start of the culture was taken as synthesis. The results showed that bile acid synthesis was linear up to 24h of incubation, at a rate of 20nmol/g hepatocytes per hour, and that 85% of the newly synthesized bile acid was cholic acid. The bile acid synthesized was mainly conjugated with taurine. These results suggest that isolated hepatocytes cultured in the way described could be a useful in vitro model for the study of bile acid synthesis.