Dog Hepatocytes Research Articles

In vivo and in vitro induction of cytochrome P450 enzymes in beagle dogs.

The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely beta-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n = 3) with beta-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with beta-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6beta-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.

Transduction of hepatocytes after neonatal delivery of a Moloney murine leukemia virus based retroviral vector results in long-term expression of beta-glucuronidase in mucopolysaccharidosis VII dogs.

The use of Moloney murine leukemia virus (MLV)-based retroviral vectors (RV) can result in stable in vivo expression in the liver, but these vectors only transduce replicating hepatocytes. As newborn animals exhibit rapid growth, we evaluated the ability of MLV-based RV to transduce hepatocytes in neonatal dogs. I.v. injection of a beta-galactosidase-expressing RV at 3 days after birth resulted in transduction of 9% of hepatocytes. Prior treatment with human hepatocyte growth factor at 2.5 mg/kg did not increase transduction. Although cells from the spleen were also transduced with moderate efficiency, cells from other organs were not. Neonatal dogs with mucopolysaccharidosis VII (MPS VII) received an i.v.injection of an RV containing the canine beta-glucuronidase (cGUSB) cDNA. At several months after transduction, clusters of hepatocytes that expressed high levels of cGUSB were present in the liver, which probably derived from replication of transduced hepatocytes. At 6 months after transduction, serum GUSB levels were 73% that of homozygous normal dogs and were 34% of the peak values observed at 1 week. We conclude that neonatal delivery of an MLV-based RV results in stable transduction of hepatocytes in dogs. This approach could result in immediate correction in patients with an otherwise-lethal genetic deficiency.

Species comparison in P450 induction: effects of dexamethasone, omeprazole, and rifampin on P450 isoforms 1A and 3A in primary cultured hepatocytes from man, Sprague–Dawley rat, minipig, and beagle dog

Induction of P450 isoforms 1A (CYP1A) and 3A (CYP3A) by model inducers dexamethasone, omeprazole and rifampin was evaluated in primary cultured hepatocytes from man and laboratory animals. Inducer-specific species-differences were observed. Results with human hepatocytes from six human donors consistently show that both rifampin and dexamethasone were inducers of CYP3A activity (measured as testosterone 6β-hydroxylase activity), with rifampin being more potent. Conversely, in rat hepatocytes, dexamethasone was a potent CYP3A inducer while rifampin was not an inducer. Rifampin but not dexamethasone induced CYP3A in minipig and beagle dog hepatocytes. Omeprazole was a potent inducer of CYP1A activity (measured as ethoxyresorufin- O-deethylase activity) in human, beagle dog and minipig hepatocytes, and not an inducer in rat hepatocytes. The species-differences observed suggest that human hepatocytes represent the most appropriate preclinical experimental system for the evaluation of P450 induction in human.

Conditions Required for a Hybrid Artificial Liver Support System using a PUF/Hepatocyte-Spheroid Packed-Bed Module and it's use in Dogs with Liver Failure

We studied the effects of a hybrid artificial liver support system we developed on dogs with hepatic failure. The system consisted of a multi-channel polyurethane foam packed-bed culture module, including primary dog hepatocyte spheroids. Blood ammonia was well metabolized by 20 g hepatocytes, but the other functions such as glucose concentration, total bile acid concentration, and survival time required 30 g hepatocytes to improve conditions. We found that we should use a culture substratum that easily forms spheroids, and that an artificial liver module should be used as soon as possible after spheroid formation by hepatocytes in the module.

Development of a Hybrid Artificial Liver using a Polyurethane Foam/Hepatocyte-Spheroid Packed-Bed Module

Primary dog hepatocytes spontaneously formed spheroids in the pores of polyurethane foam (PUF) within 1-2 days of stationary culture. The spheroids, about 100-150 microm in diameter, partly attached to the surface and immobilized inside these pores. The lidocaine disappearance rate decreased to about 4 microg/10(5) viable cells/day for 10 days, while in the PUF/spheroid culture the rate was maintained at almost the initial level of 8 microg/10(5) viable cells/day for 10 days. Then, two scales of PUF packed-bed modules were designed. A small module (PUF volume; 14.5 cm3) was used for in vitro culture to investigate optimum culture conditions, and a large module (PUF volume; 300 cm3) was designed for dog experiments. Hepatocytes inoculated in these modules also formed spheroids and maintained almost the same activity of albumin secretion rate (111 microg/cm3 PUF/day in the small module and 87.7 microg/cm3 PUF/day in the large module). These results indicate that the PUF packed-bed module containing hepatocyte-spheroids is promising as a hybrid artificial liver.

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 dexamethasonepretreated 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.

Combining in vitro and in vivo pharmacokinetic data for prediction of hepatic drug clearance in humans by artificial neural networks and multivariate statistical techniques.

Several statistical regression models and artificial neural networks were used to predict the hepatic drug clearance in humans from in vitro (hepatocyte) and in vivo pharmacokinetic data and to identify the most predictive models for this purpose. Cross-validation was performed to assess prediction accuracy. It turned out that human hepatocyte data was the best predictor, followed by rat hepatocyte data. Dog hepatocyte data and dog and rat in vivo data appear to be uncorrelated with human in vivo clearance and did not significantly contribute to the prediction models. Considering the present evaluation, the most cost-effective and most accurate approach to achieve satisfactory predictions in human is a combination of in vitro clearances on human and rat hepatocytes. Such information is of considerable value to speed up drug discovery. This study also showed some of the limitations of the approach related to the size of the database used in the present evaluation.

Characterization of drug metabolizing activities in pig hepatocytes for use in bioartificial liver devices: comparison with other hepatic cellular models

Background/Aims: The pig is considered the best donor of hepatocytes for bioartificial liver devices, but little is known about the metabolic capability of pig hepatocytes. Therefore, we have evaluated drug metabolizing activities in pig hepatocytes and liver microsomes and compared the results with those of man and other animal hepatic cellular models that are potential sources of cells for bioreactors, such as rat, rabbit and dog hepatocytes and hepatoma cell lines. Methods: Total cytochrome P450 levels, six phase 1 activities representative of the most relevant cytochrome P450 enzymes (7-ethoxycoumarin O-deethylase, 7-ethoxy-, 7-methoxy- and 7-benzoxyresorufin O-dealkylases, coumarin 7-hydroxylase and pnitrophenol hydroxylase), two phase 2 activities (glutathione S-transferase and UDP-glucuronyltransferase) and CYP-dependent regioselective testosterone metabolism were evaluated in in vitro models of different species. Results: The pattern of specific cytochrome P450 activities and the metabolic profile of testosterone in intact hepatocytes were essentially the same as those measured in liver microsomes. Relatively low ethoxy-, methoxy-, and benzoxyresorufin O-dealkylation rates were found in pig liver microsomes and hepatocytes as compared to hepatic in vitro human models. However, in contrast with the other species studied, stereoselective testosterone oxidation profiles were practically identical in human and pig models. Finally, the metabolic capability of hepatoma cell lines was very limited in comparison with that of hepatocytes. Conclusions: Pig hepatocytes are able to maintain in culture the phase 1 and phase 2 activities found in liver microsomes. The high metabolic similarities found between pig and human hepatocytes lend support to the use of pig hepatocytes in bioartificial liver devices.

An in vitro model for peroxisome proliferation utilizing primary hepatocytes in sandwich culture

Peroxisome proliferators comprise a group of structurally diverse chemicals which share as a common biologic effect the induction of peroxisomal fatty acid degrading enzymes. Concomitantly, the number and size of peroxisomes within hepatocytes increases. Following chronic administration some peroxisome proliferators act as non-genotoxic hepatocarcinogens in susceptible species such as rodents. To establish an in vitro model for the toxicological investigation of peroxisome proliferation, primary hepatocytes of rats, dogs and humans were cultivated in an organotypic cell culture model (sandwich model). By employing a panel of diverse compounds in this model a graded response was observed in the induction of carnitine acetyl transferase (CAT), the activity of which was determined as an endpoint. The following results were obtained in the order of decreasing inducing potential for rat hepatocytes: FOE 3798>nafenopin>fenofibrate (<clofibrate>ciprofibrate>bezafibrate ⪢ DEHP≈ETYA>DEHA. Induction of CAT activity was generally higher than reported in earlier cell culture systems, probably reflecting the effect of the extracellular matrix provided by the collagen gel sandwich. In parallel, transcription of the rat CYP4A1 gene was induced by a similar order of magnitude as measured by TaqMan RT-PCR. In accordance with literature data, human and dog hepatocytes did not display such a strong and graded response but rather were not susceptible to this effect. In addition, 3H-thymidine incorporation data demonstrated that nafenopin was able to induce DNA synthesis in rat hepatocytes whereas it did not in human hepatocytes.

Utility of hepatocytes to model species differences in the metabolism of loxtidine and to predict pharmacokinetic parameters in rat, dog and man

1. The metabolism of loxtidine (1-methyl-5-[3-[3-[(1-piperidinyl) methyl] phenoxy] propyl] amino-1H-1,2,4-triazole-3-methanol) was studied in freshly isolated rat, dog and human hepatocytes. Metabolism in vitro was comparable with previously available in vivo data in all three species with the marked species differences observed in vivo being reproduced in the hepatocyte model. 2. The major route for the metabolism of loxtidine by rat hepatocytes was Ndealkylation to form the propionic acid and hydroxymethyl triazole metabolites. A minor metabolic route was the oxidation of loxtidine to a carboxylic acid metabolite. The major route of metabolism for loxtidine in dog hepatocytes was glucuronidation with oxidation to the carboxylic acid metabolite being of minor importance. Incubation of loxtidine with human hepatocytes resulted in the drug remaining largely unchanged but with the carboxylic acid metabolite being produced in minor amounts. 3. In vitro studies were undertaken with rat, dog and human hepatocytes to determine the Michaelis-Menten parameters Vmax and Km for the sum of all the metabolic pathways. These kinetic parameters were used to calculate the intrinsic clearance of loxtidine. Using appropriate scaling factors, the predicted in vivo hepatic clearance was then calculated. The predicted intrinsic clearances were 51.4 ± 12.4, 8.0 ± 0.8 and 1.0 ± 0.6 ml/min/kg for rat, dog and human hepatocytes respectively. These data were then used to calculate hepatic clearances of 24.5, 3.1 and 0.2 ml/min/kg for rat, dog and man respectively. 4. In vivo hepatic and intrinsic clearances for loxtidine were determined in rat, dog and human volunteers. The hepatic clearances of loxtidine were 26.6, 6.6 and 0.4 ml/min/kg in rat, dog and man respectively and intrinsic clearances were 58.5, 18.6 and 2.0 ml/min/kg in rat, dog and man respectively. 5. The present studies demonstrate that the hepatocyte model may be a valuable in vitro tool for predicting both qualitative and quantitative aspects of the metabolism of a drug in animals and man at an early stage of the drug development process.

The formation of a spheroid of primary hepatocytes and the expression of liver-specific functions depend on the characteristics of polyurethane foam

Rapid formation of spherical multicellular aggregates (spheroids) of hepatocytes and expression of liver-specific functions are important in developing a hybrid artificial liver. The relation between these points and the surface characteristics of the culture substratum are investigated in this article. Polyurethane foam (PUF) was used as a culture substratum for hepatocytes. Rat, dog, and porcine primary hepatocytes spontaneously formed a spheroid within 1.5 days in the pores of a hydrophilic PUF with a contact angle of 53.4±2.7°. Rat and dog primary hepatocytes formed a spheroid within 3 and 7 days of culture, respectively, in hydrophobic PUF with a contact angle of about 100°. The rates of albumin secretion and ammonia metabolism at 5 days of culture of porcine hepatocytes were 31 μg of albumin/106 nuclei/day and 0.018 μmol of NH3/106 nuclei/h, respectively, about 2 times higher than the values with hydrophobic PUF. It is very important to control the hydrophilicity of the PUF surface to develop an effective artificial liver module.

The Coumarin 7-Hydroxylation Microassay in Living Hepatic Cells in Culture

Coumarin 7-hydroxylation was evaluated in hepatic cells from various species, cultured in 96-well plates. This microassay involved incubating living cultured cells with the substrate, followed by fluorimetric quantification of the product released into the culture supernatant, after hydrolysis of the conjugates of 7-hydroxycoumarin that were formed. Fluorescence was measured directly in the wells by using a microplate fluorescence reader. The major advantages of this technique are its simplicity and automation, the small number of cells required, the reduction in sample handling and assay time, and the possibility of performing repeated assays with the same cell monolayer, since no injury to cells is detectable during the assay. By using this microassay, it was shown that human hepatocytes hydroxylated coumarin at higher rates than did rabbit, dog or rat hepatocytes, and that no appreciable metabolic activity was observed in hepatoma cells (Hep G2 and FaO). In addition, methoxsalen was found to be a potent inhibitor of cytochrome P4502A6 activity in living human hepatocytes.

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.

Mepanipyrim induces fatty liver in rats but not in mice and dogs.

Mepanipyrim, a new fungicide, was administered orally to rats, mice and dogs for 13 weeks to clarify its toxic profiles. Hepatotoxicities were observed characteristically in these species with high concentrations of mepanipyrim; more than 200 ppm in rats, more than 1,000 ppm in mice, and more than 50 mg/kg/day in dogs. In rats, obvious fatty vacuolation in the perilobular hepatocytes and changes in the serum-lipid concentrations such as total cholesterol (TC), triglyceride (TG), phospholipid (PL) and non-esterified fatty acid (NEFA) were observed. This fatty liver appeared to be based on the alteration of lipid metabolism. In contrast, no remarkable changes were observed in mice and dogs except for an enhancement of anisonucleosis in mice or a lipofuscin deposition in Kupffer cells and hepatocytes in dogs. It appeared that there were species differences in the hepatotoxicities of mepanipyrim.

Phenobarbital-Induced Inclusion Bodies in Dog Hepatocytes.

Phenobarbital-Induced Inclusion Bodies in Dog Hepatocytes.

Formation of a spherical multicellular aggregate (spheroid) of animal cells in the pores of polyurethane foam as a cell culture substratum and its application to a hybrid artificial liver

Monkey kidney cells (Vero), human embryonic kidney cells (293), human liver cells (PLC/PRF/5), and primary rat, dog, and porcine hepatocytes formed spherical multicellular aggregates (spheroids) in the pores of polyurethane foam which was used as a cell culture substratum. These spheroids of various cell types express high cell activity for a long period. A practical hybrid artificial liver support system composed of a multi-capillary polyurethane foam packed-bed type cell culture module including primary hepatocyte spheroids was developed. The success of the system is indicated by an 80% recovery rate in hepatic failure rats which died in control experiments.

Tetracycline-induced steatosis in primary canine hepatocyte cultures.

Primary hepatocyte cultures prepared from male beagle dog liver were used to determine susceptibility of the canine liver to tetracycline-induced steatosis. The effects of the drug on mitochondrial lipid metabolism and intracellular triglyceride accumulation were monitored at the same time that steatosis was detected by light microscopy and quantitated using lipid-specific stains. Exposure of primary canine hepatocyte cultures to tetracycline for 24-48 h resulted in concentration-dependent, significant increases in the Oil Red O-stained lipid inclusions. Microscopic examination of the total stained areas suggested that increases over control levels were due primarily to the increase in the size of the lipid inclusions rather than in the number. Biochemical analyses for triglyceride content and histological staining with Nile red, another neutral lipid-specific dye, confirmed a specific increase in intracellular triglyceride following a 24-h exposure to noncytotoxic levels of tetracycline beta-oxidation studies based on the oxidation of [14C]palmitic acid or [14C]palmitoyl carnitine demonstrated a concentration-dependent inhibition of mitochondrial but not peroxisomal beta-oxidation in hepatocytes after a 24-h exposure to tetracycline. In vitro incubation of tetracycline with mitochondria isolated from dog liver showed similar concentration-dependent inhibition. This study clearly indicates that the canine hepatocyte is susceptible to tetracycline-induced steatosis. Triglyceride accumulation was concomitant with the inhibition of mitochondrial lipid metabolism, indicating that this is a primary mechanism leading to steatosis in dog hepatocytes following tetracycline exposure.

Effect of Thromboxane A&lt;sub&gt;2&lt;/sub&gt; Analog and Prostaglandin l&lt;sub&gt;2&lt;/sub&gt; Analog on the Isolated Hepatocytes after Extensive Hepatectomy

We evaluated the effects of TxA2 analog (STA2), TxA2 inhibitor (OKY-046) and PGI2 analog (OP-2507) on the dog hepatocytes isolated after 84% hepatectomy. Ce

Increase of cytochrome P-450 1A and glutathione transferase transcripts in cultured hepatocytes from dogs, monkeys, and humans after cryopreservation

The study was designed to investigate the effects of phenobarbital (PB), 3-methylcholanthrene (3-MC), and oltipraz (OPZ), a synthetic derivative of 1,2-dithiole-3-thione, on the levels of cytochrome P450 1A1/2 and gluthathione transferase (GST) mRNAs in both fresh and cryopreserved human, monkey, and dog hepatocytes in primary culture. GST alpha mRNAs were demonstrated in liver parenchymal cells from the three species: after 4 days of culture, their basal levels were decreased, but were strongly higher in PB- and OPZ-treated cells from the three species. In contrast 3-MC was mostly effective on human hepatocytes. The increased levels of GST alpha mRNAs in the presence of PB or OPZ were not observed in all cell populations. GST mu mRNAs, which were detected in both dog and monkey hepatocytes, were induced only in the presence of OPZ. GST pi mRNAs were expressed in dog hepatocytes but did not respond to any of the inducers. In all cases, similar effects were observed in fresh and thawed hepatocytes. Similarly, CYP1A1/2 transcripts were induced by 3-MC in both fresh and cryopreserved cells from the three species but also after OPZ treatment for monkey hepatocytes. These findings demonstrate that enzymes which play a major role in bioactivation/detoxication of xenobiotics remain expressed and inducible in hepatocytes from various species after cryopreservation and thawing.

Interspecies differences in glutathione-dependent detoxication of hydroperoxides in short-term cultures of hepatocytes

Whether the glutathione-dependent detoxication pathway of hydroperoxides is functionally expressed in vitro is not known. The major components of the system were studied in freshly isolated hepatocytes and their short-term primary monolayer cultures. The hepatocytes were derived from human organ donors, Sprague-Dawley rats, beagle dogs and cynomolgus monkeys. Species differences exist in the intracellular (reduced) glutathione (GSH) content of freshly isolated hepatocytes. In particular, the GSH content of rat hepatocytes is small in comparison with that in human cells. Monkey hepatocytes displayed the highest levels, followed by human, dog and rat cells. After 24 hr in culture, the GSH content of the hepatocytes increased, in all the species studied. With respect to the glutathione reductase activity, freshly isolated human hepatocytes showed a level that was half that found for monkeys and rats and was approximately 1.5-fold of the activity measured in dog hepatocytes. These differences were maintained during short-term culture. When glutathione peroxidase activities were measured, using hydrogen peroxide or tertiary butylhydroperoxide as substrates, the highest values were seen for rat hepatocytes followed by human, monkey and dog cells. Thus, it is clear that hepatocytes of different species have different GSH-dependent detoxication capacities.